Cleavage and polyadenylation complex of precursor mrna

ABSTRACT

The present invention relates to novel components of the cleavage/polyadenylation machinery of precursor mRNA as well as to the complex containing the new components and its use. The complex is obtained by using one component thereof as a bait and isolating a highly organized complex consisting of at least 13 distinct proteins.

1. FIELD OF THE INVENTION

[0001] The present invention relates to components of thecleavage/polyadenylation machinery of pre-cursor mRNA, the completeprotein complex, uses of said components and complex as well as tomethods of preparing same.

2. BACKGROUND OF THE INVENTION

[0002] Polyadenylation of precursor mRNA (pre-mRNAs) is an obligatorystep in the maturation of most eukaryotic transcripts. The addition ofpoly(A) (polyadenosine) tails promote transcription termination andexport of the mRNA from the nucleus. Furthermore, the poly(A) tails havethe function to increase the efficiency of translation initiation and tohelp to stabilize mRNAs. Polyadenylation occurs posttranscriptionally inthe nucleus of eukaryotic cells in two tightly coupled steps: theendonucleolytic cleavage of the precursor and the addition of a poly(A)tail.

[0003] In the yeast Saccharomyces cerevisiae, the pre-mRNA 3′-endprocessing signals are not as well conserved, as in mammalian cells (seebelow). In addition to the cleavage and polyadenylation site, twocis-acting elements, called the efficiency element and the positioningelement, are found upstream of the cleavage site. Efficiency elementscontain the sequence UAUAUA (or close variants thereof) and are oftenrepeated. The sequence AAUAAA and several related sequences can functionas a positioning element.

[0004] Fractionation of yeast extracts led to the separation of proteinfactors that are required for mRNA 3′-end formation in vitro. Thecleavage reaction requires cleavage factors I and II (CF I and CF II),whereas polyadenylation involves CF I, polyadenylation factor I (PF I)and poly(A) polymerase (Pap1).

[0005] CF I can be separated into two activities, CF IA and CF IB. CF IAis needed for both processing steps and is a heterotetrameric proteinwith subunits of 38, 50, 70 and 76 kDa that are encoded by the RNA5,CLP1, PCF1 1 and RNA14 genes. Rna14 shares significant sequencesimilarity to the 77 kDa subunit of mammalian cleavage stimulationfactor (CstF) and Rna15 contains a RNA-binding domain homologous to thatof the 64 kDa subunit of CstF.

[0006] In addition to the above mentioned four CFI subunits, Pab1(poly(A) binding protein) was identified in purified CFI fractions. Bothbiochemical and genetic data indicate an involvement of Pab1 in poly(A)length control. CF IB consists of a single protein called Nab4/Hrp1 andis required for cleavage site selection and polyadenylation. Amultiprotein complex which has CFII-PF I (=CPF) activity consists ofnine polypeptides: Pap1 (poly(A) polymerase), Pta1, Pfs1, Pfs2, Fip1,Cft1/Yhh1, Cft2/Ydh1, Ysh1/Brr5, and Yth1. Pap1, a 64 kDa protein, wasthe first component of the yeast 3′-end formation complex to be purifiedto homogeneity. Pta1 is a 90 kDa protein which is required for bothcleavage and polyadenylation of mRNA precursors. Pfs2 is a 53 kDaprotein that contains seven WD40 repeats. Pfs2 has been shown todirectly interact with subunits of CFII-PF1 and CFIA and is thought tofunction in the assembly and stabilization of the 3′-end processingcomplex. Fip1 has been demonstrated to physically interact with Pap1,Yth1 and Rna14 and it is believed that it tethers Pap1 to its substrateduring polyadenylation. Cft1/Yhh1, Cft2/Ydh1, Ysh1/Brr5, and Yth1 arethe counterparts of the four subunits of the mammalian cleavage andpolyadenylation specificity factor, CPSF160, CPSF100, CPSF73 and CPSF30,respectively.

[0007] Furthermore TIF4632 has been found to interact with Pab1 (seeTable 1)

[0008] For the mammalian system, various data have been presented whichhave given evidence both for a conserved mechanism and also showed somedifferences between the yeast and the mammalian structures.

[0009] The composition and function of the mammalian complex based onthe data to date is as follows:

[0010] The cleavage and polyadenylation factor (CPSF) is composed of 4subunits: CPSF160 (involved in mRNA and poly(A) polymerase (PAP)binding), CPSF100, CPSF 73 and CPSF30 (involved in mRNA and PABIIbinding).

[0011] CPSF binds the AAUAAA hexanucleotides. CPSF links the mRNA 3′-endprocessing to the transcription. CPSF exists as a stable complex withthe transcription factor TFIID complex. The 160 kDa subunit of CPSFbinds to several hTAFII. TFIID recruits CPSF to the RNA polymerase IIpre-initiation complex. Upon transcriptional activation CPSF dissociatesfrom TFII and associates with the elongating RNA pol II (CTDcarboxy-terminal domain of the largest subunit of the RNA polymeraseII). CPSF is thought to travel with RNA pol II until they reach thepolyadenylation site, where CPSF can bind the AAUAAA element. CPSF isrequired for the termination of transcription.

[0012] The interaction between CPSF and the AAUAAA element is weak andnot so specific. The binding of CPSF to the hexanucleotide is greatlyenhanced by a 2nd component of the poly-adenylation machinery, thecleavage stimulation factor (CstF), which binds the G-U rich motif. CstFalso binds the RNA pol II through its 50 kDa subunit (CstF50).Furthermore, CstF50 binds another component of the transcriptionalmachinery: BRCA1 associated RING domain protein (BARD1). BARD1 alsointeracts with RNA pol II. BARD1-CstF50 interaction inhibitspolyadenylation in vitro and may prevent inappropriate mRNA processingduring transcription. CstF is composed of 3 subunits: CstF64 (binds mRNAand symplekin (yeast homolog: Pta1), CstF77 (binds CPSF160, CstF64,CstF50) and CstF50 (binds RNA pol II and BARDL). The co-operativebinding of CPSF and CstF to the polyadenylation site forms a ternarycomplex, which functions to recruit the other components of thepolyadenylation machinery to the cleavage site: the two cleavage factors(CFIm and CFIIm) and the poly(A) polymerase (PAP).

[0013] CFIm is an heterodimer of 4 subunits 72, 68, 59, 25 components:one essential, CFIImA and one stimulatory, CFIIB. CFIImA containshPCF11p and hClp1p (binds cPSF and CF I). CF IImB contains no factorspreviously shown to be involved in 3′-end processing and may be a new3′-end processing factor. Although the identity of the proteins thatperform the cleavage step is still unknown, it is well established thatboth CFIm and CFIIm are required. The reaction products of the cleavagesuggest that a metal ion is involved. Surprisingly, PAP (but not itscatalytic activity) is required for the cleavage.

[0014] After the cleavage step CstF, CFIm and CFIIm are dispensable. PAPbound to CPSF (through its 160 kD subunit) can start polyadenylating thecleaved 3′-end, but at that step, the process is very inefficient. Thepoly(A) binding protein II (PAB II) can bind the nascent poly(A) chainas soon as it reaches a minimal length of 10 poly(A). PAB II alsointeracts with the CPSF30. The binding of PAB II greatly stabilizes PAPat the 3′-end of the mRNA, supporting the progressive synthesis of along poly(A) tail. In the nucleus, the length of the poly(A) tail isrestricted to about 250 poly(A). This size restriction is probablyachieved through stoichiometric binding of multiple PAB II. It is notyet known how the incorporation of a certain amount of PAB II in thecomplex terminates processive elongation.

[0015] CstF is part of the mammalian 3′-end processing complex and is aheterotrimeric protein with subunits of 77, 64 and 50 kDa. CstF-50 hasbeen shown to interact with the BRCA1-associated protein BARD1 and thisinteraction suppresses the nuclear mRNA polyadenylation machinery invivo. In a recent study it was found that treatment of cells with DNAdamage-inducing agents causes a transient, but specific, inhibition ofmRNA 3′-end processing in cell extracts. This inhibition reflects theBARD1/CstF interaction and involves enhanced formation of aCstF/BARD1/BRCA1 complex. A tumor-associated germline mutation in BARD1decreases binding to CstF-50 and renders the protein inactive inpolyadenylation inhibition. These results support the existence of alink between mRNA 3′-end formation and DNA repair/tumor suppression. Thein vivo function of these interactions may be to inhibit the cleavageand polyadenylation of pre-mRNAs on polymerase molecules that arestalled at sites of DNA repair.

[0016] Cleavage stimulation factor (CstF) is one of the multiple factorsrequired for mRNA polyadenylation in mammalians. CstF-64 may play a rolein regulating gene expression and cell growth in B cells. Theconcentration of one CstF subunit (CstF-64) increases during activationof B cells, and this is sufficient to switch IgM heavy chain mRNAexpression from membrane-bound to secreted form. Reduction in CstF-64causes reversible cell cycle arrest in G0/G1 phase, while depletionresults in apoptotic cell death.

[0017] In contrast to what is observed in yeast, the sequence elementsin mammals, which specify the site of cleavage and polyadenylation,flank the site of endonucleolytic attack. One is the hexanucleotideAAUAAA found 10-30 bases upstream of the cleavage/polyadenylation site.The second is a G-U-rich motif located 20-40 bases downstream of thecleavage/polyadenylation site. These two elements and their spacingdetermine the site of cleavage/polyadenylation and also the strength ofthe polyadenylation signal.

[0018] Some other elements, like sequences upstream of the AAUAAA(upstream sequence elements, USEs) play regulatory roles.

[0019] A schematic presentation of the motifs underlying mammalianpolyadenylation and yeast polyadenylation are shown in FIG. 1. A reviewon the formation of mRNA 3′-ends in eukaryotes is given in Zhao, Hymanand Moore in Microbiology and Molecular Biology Reviews, 1999, pp.405-445. A comparison of mammalian and yeast pre-mRNA 3′-end processingis also given in Keller and Minvielle-Sebastia in Nucleus and geneexpression in Current Opinion in Cell Biology, 1997, Vol. 9, pp.329-336.

[0020] There are diseases which involve defects in the function of thepolyadenylation machinery.

[0021] Many viruses interact directly with components of the mRNAprocessing machinery.

[0022] The herpes simplex virus type 1 (HSV-1) immediate early (alpha)protein ICP27 is an essential regulatory protein that is involved in theshutoff of host protein synthesis. It affects mRNA processing at thelevel of both polyadenylation and splicing. During polyadenylation,ICP27 appears to stimulate 3′ mRNA processing at selected poly(A) sites.The opposite effect occurs on host cell splicing. That is, during HSV-1infection, an inhibition in host cell splicing requires ICP27expression. This contributes to the shutoff of host protein synthesis bydecreasing levels of spliced cellular mRNAs available for translation. Aredistribution of splicing factors regulated by ICP27 has also beenseen.

[0023] Epstein-Barr virus BMLF1 gene product EB2 seems to affect mRNAnuclear export of intronless mRNAs and pre-mRNA 3′ processing. EB2contains an Arg-X-Pro tripeptide repeated eight times, similar to thatdescribed as an mRNA-binding domain in the herpes simplex virus type 1protein US11.

[0024] Interestingly, both viruses have been found to precede the onsetof lymphomas. Influenza A virus NS1A protein binds the 30 kDa subunit ofthe cleavage and polyadenylation specificity factor (CPSF), NS1 protein(NS1A protein) via its effector domain targets the poly(A)-bindingprotein II (PABII) of the cellular 3′-end processing machinery. In vitrothe NS1A protein binds the PABII protein, and in vivo causes PABIIprotein molecules to relocalize from nuclear speckles to a uniformdistribution throughout the nucleoplasm. In vitro the NS1A proteininhibits the ability of PABII to stimulate the processive synthesis oflong poly(A) tails catalyzed by poly(A) polymerase (PAP). Suchinhibition also occurs in vivo in influenza virus-infected cells.Consequently, although the NS1A protein also binds the 30 kDa subunit ofthe cleavage and polyadenylation specificity factor (CPSF), 3′ cleavageof some cellular pre-mRNAs still occurs in virus-infected cells,followed by the PAP-catalyzed addition of short poly(A) tails.Subsequent elongation of these short poly(A) tails is blocked becausethe NS1A protein inhibits PABII function. The NS1 effector domainfunctionally interacts with the cellular 30 kDa subunit of CPSF, anessential component of the 3′ end processing machinery of cellularpre-mRNAs.

[0025] Metachromatic leukodystrophy (MLD) is a lysosomal storagedisorder caused by the deficiency of arylsulfatase A (ASA). Asubstantial ASA deficiency has also been described in clinically healthypersons, a condition for which the term pseudodeficiency was introduced.The mutations characteristic for the pseudodeficiency (PD) allele havebeen identified. Sequence analysis revealed two A-G transitions. One ofthem changes the first polyadenylation signal downstream of the stopcodon from AATAAC to AGTAAC. This causes a severe deficiency of a2.1-kilobase (kb) mRNA species. The deficiency of the 2.1-kb RNA speciesprovides an explanation for the diminished synthesis of ASA seen inpseudodeficiency fibroblasts.

[0026] MLD patients have been identified who are homozygous for theASA-PD allele and it is thought that the allele might play a role in thedevelopment and progression of disease.

[0027] There is a tight link between cell cycle control andpolyadenylation machinery suggesting an important role of this machineryin the development of cancer. Cyclin-dependent enzymes seem to regulatethe activity of the polyadenylation machinery. The amounts of somefactors of the mRNA 3′ processing machinery (CstF) increase inmitotically active cells in phases of the cell cycle preceding DNAsynthesis. The amount of the 64-kDa subunit CstF-64 increases 5-foldduring the G0 to S phase transition and concomitant proliferationinduced by serum in 3T6 fi-broblasts. The increase in CstF-64 isassociated with the G0 to S phase transition. Cdc2-cyclin Bphosphorylates PAP at the Ser-Thr-rich region.

[0028] However, as it seems now, most diseases associated with defectsin mRNA processing are caused by mutations in cis-acting elements thatdisrupt sequences essential for pre-mRNA splicing. These can becanonical sequences at the intron-exon border or located within an exon.They directly affect the expression of a single mutated gene.Approximately 15% of the nucleotide substitutions that cause humandiseases disrupt pre-mRNA splicing. Thus these diseases do not seem tobe directly caused by alterations in thepolyadenyation/cleavage-machinery.

[0029] However, since recently evidence for a number ofinterrelationships between polyadenylation/cleavage and splicing isaccumulating (for review see Zhao, Hyman and Moore in Microbiology andMolecular Biology Reviews, 1999, pp. 405-445), it might very well bethat alterations in the 3′-end processing machinery contribute to theetiology of these diseases.

[0030] Examples of diseases caused by incorrect splicing are mentionedbelow:

[0031] Amyotrophic lateral sclerosis (ALS) is a neurodegenerativedisease involving degeneration of cortical motor neurons andspinal/bulbar motor neurons. In the sporadic form of the disease, theneuron degeneration is caused by excessive extracellular glutamate. Theglutamate transporter functional in the CNS is the astrocyte EAAT2 whichis altered in ALS. The pre-mRNA for EAAT2 is aberrantly spliced in thebrain regions affected. The reason for this is still unknown, but thedefect lies probably in one or a few auxiliary splicing factors thatregulate the splicing of a sub-set of pre-mRNA in these cells. Thefactors have not yet been identified.

[0032] The human papillomavirus (HPV) E2 protein plays an important rolein transcriptional regulation of viral genes as well as in viral DNAreplication. HPV-5 (an EV epidermodysplasia verruciformis-HPV) proteincan specifically interact with cellular splicing factors including a setof prototypical SR proteins and two snRNP-associated proteins (Lai, Tehet al. 1999, J. Biol. Chem. 274, pages 11832-41). Interestingly allthese three viruses have been associated with cancer progression.Papillomavirus infection precedes cervical cancer, whereas EBV and HSV-8have been described in association with lymphomas.

[0033] In hepatocellular carcinoma, there is a defect in mRNA splicing.In this disease, there are anti-nuclear antibodies to a 64 kD protein,which has splicing factor motifs. A defect in the regulatory subunit 3of the protein phosphatase 1(PP1) has been found in haematologicalmalignancies and in lung, ovarian, colorectal and gastric cancers. LowPP1 activity has been observed also in acute myelogenous leukaemia.

[0034] Heterogeneous nuclear ribonucleoproteins (hnRNP) associate withpre-mRNA and have a role in RNA processing and splice site selection.HnRNP A2 shows a marked overexpression in lung cancer and brain tumoursand has thus been used as a biomarker for these tumor types.

[0035] The development of antinuclear antibodies (ANA) in malignancieshas been described but its mechanism is still not understood. A greatdiversity of ANA specificities is found in hepatocellular carcinoma. Inhepatoma sera antibodies co-localize with non-snRNP splicing factorSC35, suggesting that the antigenic targets might be involved in mRNAsplicing. Hepatocellular carcinoma has a significantly higher frequencyof ANA than chronic hepatitis C, chronic hepatitis B, alcoholic livercirrhosis or healthy donors.

[0036] In some autoimmune diseases, a possible link has been detected toa preceding virus infection, like Epstein-Barr virus in SLE. Furthermoreit seems that even vaccination is potentially dangerous: a candidate forcytomegalovirus CMV vaccine is glycoprotein gB (UL55). Immunization withan adenovirus-gB construct (Ad-gB) not only induces a significantanti-viral response, but a significant IgG auto-antibody response(p>0.005) to the U1-70 kDa spliceosome protein. Auto-antibodies to U1-70kDa are part of the anti-ribonucleoprotein response seen in systemiclupus erythematosus and mixed connective tissue disease.

[0037] At least two molecules which are also part of the complex areknown to be inhibited by natural toxins or treatment against variousdiseases.

[0038] Protein phosphatase 1 is inhibited by several natural producttoxins.

[0039] The marine toxins include the cyanobacteria-derived cyclicheptapeptide microcystin-LR and the polyether fatty acid okadaic acidfrom dinoflagellate sources. They bind to a common site on PP1. Thedephosphorylation of PP1 is inhibited (among other serine/threoninephosphatases PP2A, PP2B, PP2C and PP5/T/K/H ) by Fumonisin B1 (FB1), amycotoxin produced by the fungus Fusarium moniliforme. This is a commoncontaminant of corn, and is suspected to be a cause of human esophagealcancer. FB1 is hepatotoxic and hepatocarcinogenic in rats, although themechanisms involved have not been clarified.

[0040] Viral proteins are able to interfere with PP1 activity:

[0041] The transcription factor EBNA2 of the Epstein-Barr virus inducesthe expression of LMP1 onco-gene in human B-cells. EBNA2A from anEBV-immortalized B-cell line co-immunopurifies with a PP1-like protein.A PP1-like activity in nuclear extracts from EBV-immortalized B-cellline can be inhibited by a GST-EBNA2A fusion product.

[0042] Poly(A)polymerase (PAP) is affected by anticancer drugs and isinhibited by some antiviral agents.

[0043] Anticancer drugs:

[0044] Most anticancer drugs act through the mechanism of apoptosis.Apoptosis may be regulated at all levels of gene expression includingthe addition of the poly(A) tail to the 3′ end of mRNAs. Drugcombinations are more effective than single drugs and variouschemotherapeutic strategies have therefore been developed.Dimethylsulfoxide (DMSO) in combination with interferon (IFN) results inpronounced PAP dephosphorylation, activity reduction and apoptosis ofHeLa cells.

[0045] Purine and pyrimidine analogues often affect PAP activity. Theyare potentially useful agents for chemotherapy of cancer diseases. Theanticancer drugs 5-Fluorouracil (5-FU), interferon and tamoxifen mediateboth partial dephosphorylation and inactivation of poly(A) polymerase(PAP).

[0046] PAP (from isolated hepatic nuclei) is inhibited by cordycepin5′-triphosphate. The nucleoside analogue cordycepin is a therapeuticagent for TdT+(terminal deoxynucleotidyl transferase positive) leukemia.In the presence of an adenosine deaminase inhibitor, deoxycoformycin(dCF), cordycepin is cytotoxic to leukemic TdT+ cells. A cordycepinanalog of (2′-5′) oligo(A) which can be synthesized enzymatically fromcordycepin 5′-triphosphate and the core cordycepin analog can replacehuman fibroblast interferon in preventing the transformation of humanlymphocytes after infection with Epstein-Barr virus B95-8 (EBV). Thecore cordycepin analog is not cytotoxic to uninfected lymphocytes andproliferating lymphoblasts.

[0047] Not only is PAP affected by anticancer drugs, but it has apossible use as a tumor marker involved in cell commitment and/orinduction of apoptosis and could be used to evaluate tumor cellsensitivity to anticancer treatment.

[0048] Antiviral drugs:

[0049] Ara-ATP (arabinofuranosyladenosine triphosphate) is anantiherpetic drug that inhibits herpes simplex virus replication. Itinhibits poly(A) polymerase activity by competing with ATP. It blocksboth cleavage and polyadenylation reactions by interacting with theATP-binding site on poly(A) polymerase, the activity of which isessential for the cleavage reaction.

[0050] Purine and pyrimidine analogues are also used as antiviralagents. As an example, the most extensively used drug against HSV isidoxyuridine, the 5′-amino analog of thymidine.

[0051] A decrease in herpes simplex virus transcription and perturbationof RNA polyadenylation is induced by 5′-amino-5′-deoxythymidine (AdThd).

[0052] The cleavage stimulation factor (CSTF):

[0053] Treatment with hydroxyurea or ultraviolet light strongly, buttransiently, inhibits 3′ cleavage. This is accompanied by increasedamounts of a CstF/BARD1/BRCA1 complex, though the amount of theseproteins remains the same.

[0054] Despite the large body of information already available from theprior art concerning the cleavage/polyaderiylation machinery ofprecursor mRNA up to now not all components of the machinery are knownnot to speak of the composition of the complex as a whole.

3. SUMMARY OF THE INVENTION

[0055] An object of the present invention was to identify the componentsof the cleavage/polyadenylation machinery of precursor mRNA and providenew components of the cleavage/polyadenylation machinery to provide themachinery and to provide new targets for therapy.

[0056] By applying the process according to the invention to theisolation of the polyadenylation/cleavage machinery from yeast 32 newcomponents could be identified which are Act1 (SEQ ID:1), Cka1 (SEQID:7), Eft2 (SEQ ID 11), Eno2 (SEQ ID: 13), Glc7 (SEQ ID:15), Gpm1 (SEQID:17), Hhf2 (SEQ ID:21), Hta1 (SEQ ID:23), Hsc82 (SEQ ID:25), Imd2 (SEQID:27), Imd4 (SEQ ID:29), Met6 (SEQ ID:31), Pdc1 (SEQ ID:39), Pfk1 (SEQID:41), Ref2 (SEQ ID:47), Sec13 (SEQ ID:53), Sec31 (SEQ ID:55), Ssa3(SEQ ID:57), Ssu72 (SEQ ID: 59), Taf60 (SEQ ID:61), Tkl1 (SEQ ID:65),Tsa1 (SEQ ID: 67), Tye7 (SEQ ID: 69), Vid24 (SEQ ID:71), Vps3 (SEQ ID:73), Ycl046w (SEQ ID: 79), Ygr156w (SEQ ID: 81), Yhl035c (SEQ ID:83),Ykl018w (SEQ ID:85), Ylr221c (SEQ ID: 87), Yml030w (SEQ ID:91) andYor179c (SEQ ID:93).

[0057] Said object is further achieved by the characterisation ofYcl046w (SEQ ID: 79), Ygr156w (SEQ ID: 81), Yhl035c (SEQ ID:83), Ykl018w(SEQ ID:85), Ylr221c (SEQ ID: 87), Yml030w (SEQ ID:91) and Yor179c (SEQID:93) as components of the cleavage/polyadenylation machinery.

[0058] The invention thus relates to:

[0059] An isolated complex selected from complex (I) and comprising

[0060] (a) a first protein, or a functionally active fragment orfunctionally active derivative thereof, which first protein is selectedfrom the group of proteins in Table 1, column A, or a mammalian homologthereof, or a variant of said protein encoded by a nucleic acid thathybridizes to the nucleic acid of said protein or its complement underlow stringency conditions; and

[0061] (b) a second protein, or a functionally active fragment orfunctionally active derivative thereof, which second protein is selectedfrom the group of proteins in Table 1, column B, or a mammalian homologthereof, or a variant of said protein encoded by a nucleic acid thathybridizes to the nucleic acid of said protein or its complement underlow stringency conditions, wherein said first protein and said secondprotein are members of a native cellular Polyadenylation-complex, andwherein said low stringency conditions comprise hybridization in abuffer comprising 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ug/ml denatured salmonsperm DNA, and 10% (wt/vol) dextran sulfate for 18-20 hours at 40° C.,washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS for 1.5 hours at 55° C., and washing in a bufferconsisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDSfor 1.5 hours at 60° C. and a complex (II) comprising at least twosecond proteins.

[0062] Furthermore, the invention relates to an isolated complexcomprising all proteins in column C of table 1, or the mammalianhomologs of those proteins, or variants of said proteins encoded bynucleic acid that hybridises to the nucleic acid of any of said proteinsor its complements under low stringency conditions, wherein proteins aremembers of a native cellular complex, and wherein said low stringencyconditions comprise hybridization in a buffer comprising 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2%BSA, 100 ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextransulfate for 18-20 hours at 40° C., washing in a buffer consisting of2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at55° C., and washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60° C.

[0063] Furthermore, the invention relates to an isolated complex thatcomprises all but 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17,18,19,20,21,22,23,24,25,26,27 or 28 of all proteins in column C oftable 1.

[0064] Furthermore, the invention relates to the complex as describedabove comprising a functionally active derivative of said first proteinand/or a functionally active derivative of said second protein, whereinthe functionally active derivative is a fusion protein comprising saidfirst protein or said second protein fused to an amino acid sequencedifferent from the first protein or second protein, respectively.

[0065] In a preferred embodiment of the present invention, the proteincomponents of the complex are vertebrate homologs of the yeast proteins,or a mixture of yeast and vertebrate homolog proteins. In a morepreferred embodiment, the protein components of the complex aremammalian homologs of the yeast proteins, or a mixture of yeast andmammalian homolog proteins. In particular aspects,n the native componentproteins, or derivatives or fragments of the complex are obtained from amammal such as mouse, rat, pig, cow, dog, monkey, human, sheep or horse.In another preferred embodiment, the protein components of the complexare human homologs of the yeast proteins, or a mixture of yeast andhuman homolog proteins. In yet another preferred embodiment, the proteincomponents of the complex are a mixture of yeast, vertebrate, mammalianand/or human proteins.

[0066] Furthermore, the invention relates to a complex as describedaboveof claim that is involved in the 3′ end processing activity. Such acomplex might also exist as a module or subcomplex of a largerphysiological protein complex or assembly.

[0067] Furthermore, the invention relates to a complex as describedabove comprising a fragment of said first protein and/or a fragment ofsaid second protein, which fragment binds to another protein componentof said complex.

[0068] Furthermore, the invention relates to a complex as describedabove, wherein the functionally active derivative is a fusion proteincomprising said first protein or said second protein preferentiallyfused to an affinity tag or label.

[0069] It is further directed to complexes comprising a fusion proteinwhich comprises a component of the complex or a fragment thereof linkedvia a covalent bond to an amino acid sequence different from saidcomponent protein, as well as nucleic acids encoding the protein,fusions and fragments thereof. For example, the non-component proteinportion of the fusion protein, which can be added to the N-terminal, theC-terminal or inserted into the amino acid sequence of the complexcomponent can comprise a few amino acids, which provide an epitope thatis used as a target for affinity purification of the fusion proteinand/or complex.

[0070] Furthermore the invention relates to a process for processing RNAcomprising the step of bringing into contact any of the complexesdescribed above with RNA, such that RNA is processed.

[0071] Furthermore, the invention relates to an antibody or a fragmentof said antibody containing the binding domain thereof, which binds thecomplex as described above of claim and which does not bind the firstprotein when uncomplexed or the second protein when uncomplexed.

[0072] Furthermore, the invention relates to a pharmaceuticalcomposition comprising the protein complex as described above and apharmaceutically acceptable carrier.

[0073] Moreover, the present invention provides a process for theidentification and/or preparation of an effector of a compositionaccording to the invention which process comprises the steps of bringinginto contact the composition of the invention or of a component thereofwith a compound, a mixture of compounds or a library of compounds anddetermining whether the compounds or certain compounds of the mixture orlibrary bind to the composition of the invention and/or a componentthereof and/or affects the biological activity of such a composition orcomponent and then optionally further purifying the compound positivelytested as effector by such a process.

[0074] A major application of the composition according to the inventionresults in the identification of an active agent capable of bindingthereto. Hence, the compositions of the invention are useful tools inscreening for new pharmaceutical drugs.

[0075] Furthermore, the invention relates to a method for screening fora molecule that modulates directly or indirectly the function, activity,composition or formation of the complex as described above comprisingthe steps of:

[0076] (a) exposing said complex, or a cell or organism containing saidcomplex to one or more candidate molecules; and

[0077] (b) determining the amount of, the 3′ end processing activity formRNA of, or protein components of, said complex, wherein a change insaid amount, activity, or protein components relative to said amount,activity or protein components in the absence of said candidatemolecules indicates that the molecules modulate function, activity orcomposition of said complex.

[0078] Furthermore, the invention relates to a method as describedabove, wherein the amount of said complex is determined.

[0079] Furthermore, the invention relates to a method as describedabove, wherein the activity of said complex is determined.

[0080] Furthermore, the invention relates to a method as describedabove, wherein said determining step comprises isolating from the cellor organism said complex to produce said isolated complex and contactingsaid isolating complex with the substrate under conditions conducive tobinding to the complex.

[0081] Furthermore, the invention relates to a method as describedabove, wherein the protein components of said complex are determined.

[0082] Furthermore, the invention relates to a method as describedabove, wherein said determining step comprises determining whether anyof the proteins listed in column B of table 1 of said complex or themammalian homologs thereof, or variant of said proteins encoded by anucleic acid that hybridises to the nucleic acids of any of saidproteins or its complements under low stringency conditions, is presentin the complex, wherein said low stringency conditions comprisehybridization in a buffer comprising 35% formamide, 5×SSC, 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 55° C., andwashing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS for 1.5 hours at 60° C.

[0083] Furthermore, the invention relates to a method as describedabove, wherein said method is a method of screening for a drug fortreatment or prevention of diseases and disorders, preferably diseasesor disorders such as infectious diseases; viral infections such asherpes simplex infections, Epstein-Barr-infections, influenza; metabolicdisease such as metachromatic leukodystrophy; neurodegenerativedisorders such as amyotrophic lateral sclerosis and cancer.

[0084] Furthermore, the invention relates to a method for screening fora molecule that binds the complex as described above comprising thefollowing steps:

[0085] (a) exposing said complex, or a cell or organism containing saidcomplex, to one or more candidate molecules; and

[0086] (b) determining whether said complex is bound by any of saidcandidate molecules.

[0087] Furthermore, the invention relates to a method for diagnosing orscreening for the presence of a disease or disorder or a predispositionfor developing a disease or disorder in a subject, which disease ordisorder is characterized by an aberrant amount of, the 3′ endprocessing activity for mRNA biochemical activity of, or componentcomposition or formation of, the complex as described above, comprisingdetermining the amount of, the 3′ end processing activity for mRNA of,or protein components of, said complex in a sample derived from asubject, wherein a difference in said amount, activity, or proteincomponents of, said complex in an analogous sample from a subject nothaving the disease or disorder or predisposition indicates the presencein the subject of the disease or disorder or predisposition.

[0088] Furthermore, the invention relates to a method as describedabove, wherein the amount of said complex is determined.

[0089] Furthermore, the invention relates to a method as describedabove, wherein the activity of said complex is determined.

[0090] Furthermore, the invention relates to a method as describedabove, wherein said determining step comprises isolating from the cellor organism said complex to produce said isolated complex and contactingsaid isolating complex with the substrate under conditions conducive tobinding to the complex.

[0091] Furthermore, the invention relates to a method as describedabove, wherein the protein components of said complex are determined.

[0092] Furthermore, the invention relates to a method as describedabove, wherein said determining step comprises determining whether anyof the proteins listed in column B of table 1 of said complex or themammalian homologs thereof, or variant of said proteins encoded by anucleic acid that hybridises to the nucleic acids of any of saidproteins or its complements under low stringency conditions, is presentin the complex, wherein said low stringency conditions comprisehybridization in a buffer comprising 35% formamide, 5×SSC, 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 55° C., andwashing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS for 1.5 hours at 60° C.

[0093] Furthermore, the invention relates to a method for treating orpreventing a disease or disorder characterized by an aberrant amount of,the 3′ end processing activity for mRNA of, or component composition orformation of, the complex as described above, comprising administeringto a subject in need of such treatment or prevention a therapeuticallyeffective amount of one or more molecules that modulate the amount of,the 3′ end processing activity for mRNA of, or protein components orformation of, said complex.

[0094] Furthermore, the invention relates to a method as describedabove, wherein said disease or disorder involves decreased levels of theamount or activity of said complex. Furthermore, the invention relatesto a method as described above, wherein said disease or disorderinvolves increased levels of the amount or activity of said complex.

[0095] Furthermore, the invention relates to the use of a molecule thatmodulates the amount of, the 3′ end processing activity for mRNA of, orprotein components or formation of the complex as described above forthe manufacture of a medicament for the treatment or prevention of adisease or disorder, preferably diseases or disorders such as infectiousdiseases; viral infections such as herpes simplex infections,Epstein-Barr-infections, influenza; metabolic disease such asmetachromatic leukodystrophy; neurodegenerative disorders such asamyotrophic lateral sclerosis; cancer

[0096] Furthermore, the invention relates to a kit comprising in one ormore containers

[0097] (a) an isolated first protein, or a functionally active fragmentor functionally active derivative thereof selected from the proteins incolumn A of table 1 of a given complex or a mammalian homolog thereof,or a variant of said protein encoded by a nucleic acid that hybridisesto the nucleic acid of said protein or its complement under lowstringency conditions; and

[0098] (b) an isolated second protein, or a functionally active fragmentor functionally active derivative thereof selected from the proteins incolumn B of table 1 of a given complex or a mammalian homolog thereof,or a variant of said protein encoded by a nucleic acid that hybridisesto the nucleic acid of said protein or its complement under lowstringency conditions, wherein said first and said second protein aremembers of a native cellular complex, and wherein said low stringencyconditions comprise hybridization in a buffer comprising 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2%BSA, 100 ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextransulfate for 18-20 hours at 40° C., washing in a buffer consisting of2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at55° C., and washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60° C.

[0099] Furthermore, the invention relates to a kit comprising in acontainer the isolated complex as described above or the antibody asdescribed above, optionally together with further reagents and workinginstructions. The further reagents may be, for example, buffers,substrates for enzymes but also carrier material such as beads, filters,microarrays and other solid carries. The working instructions mayindicate how to use the ingredients of the kit in order to perform adesired assay.

[0100] Furthermore, the invention relates to such kits for use inprocessing of RNA and for use in the diagnosis, prognosis and screeningin or for the diseases mentioned above.

[0101] Furthermore, the invention relates to a complex as describedabove, or the antibody or fragment as described above, for use in amethod of diagnosing a disease or disorder, preferably the diseases ordisorders as mentioned above.

[0102] Furthermore, the invention relates to a method for the productionof a pharmaceutical composition comprising carrying out the method asdescribed above to identify a molecule that modulates the function,activity or formation of said complex, and further comprising mixing theidentified molecule with a pharmaceutically acceptable carrier.

[0103] Furthermore, the invention relates to a process for preparingcomplex as described above and optionally the components thereofcomprising the following steps: expressing such a protein in a targetcell, isolating the protein complex which is attached to the taggedprotein, and optionally disassociating the protein complex and isolatingthe individual complex members.

[0104] Furthermore, the invention relates to the process as describedabove characterized in that the tagged protein comprises two differenttags which allow two separate affinity purification steps.

[0105] Furthermore, the invention relates to the process as describedabove, characterized in that two tags are separated by a cleavage sitefor a protease.

[0106] Furthermore, the invention relates to a component of the saidcomplex obtainable by a process as described above.

[0107] The present invention further relates to a composition,preferably a protein complex, which is obtainable by the methodcomprising the following steps: tagging a protein as defined above, i.e.a protein which forms part of a protein complex, with a moiety,preferably an amino acid sequence, that allows affinity purification ofthe tagged protein and expressing such protein in a target cell andisolating the protein complex which is attached to the tagged protein.The details of such purification are described in WO 00/09716 and inRigaut, G. et al. (1999), Nature Biotechnology, Vol. 17 (10): 1030-1032and further herein below. The tagging can essentially be performed withany moiety which is capable of providing a specific interaction with afurther moiety, e.g. in the sense of a ligand receptor interaction,antigen antibody interaction or the like. The tagged protein can also beexpressed in an amount in the target cell which comes close to thephysiological concentration in order to avoid a complex formation merelydue to high concentration of the expressed protein but not reflectingthe natural occurring complex.

[0108] In a further preferred embodiment, the composition is obtained byusing a tagged protein which comprises two different tags which allowtwo different affinity purification steps. This measure allows a higherdegree of purification of the composition in question. In a furtherpreferred embodiment the tagged protein comprises two tags that areseparated by a cleavage site for a protease. This allows a step-by-steppurification on affinity columns.

[0109] Furthermore, the invention relates to a complex as describedabove and/or protein thereof as a target for an active agent of apharmaceutical, preferably a drug target in the treatment or preventionof disease or disorder, preferably diseases or disorders as mentionedabove.

[0110] Furthermore, the invention relates to the Ycl046w (SEQ ID: 59),Ygr156w (SEQ ID: 61), Yhl035c (SEQ ID:63), Ykl018w (SEQ ID:179), Ylr221c(SEQ ID: 67), Yml030w (SEQ ID:69), and Yor17c (SEQ ID:71), the mammalianhomologs/orthologs of said proteins and functionally active fragmentsand derivatives of said proteins and the mammalian homologs thereofcarrying one or more amino acid substitutions, deletions and/oradditions and the nucleic acid encoding said proteins or said homologs,orthologs and functionally active fragments and derivatives thereof.

[0111] Such a nucleic acid may be used for example to express a desiredtagged protein in a given cell for the isolation of a complex orcomponent according to the invention. Such a nucleic acid may also beused for the identification and isolation of genes from other organismsby cross species hybridization.

[0112] The present invention further relates to a construct, preferablya vector construct, which comprises a nucleic acid as described above.Such constructs may comprise expression controlling elements such aspromoters, enhancers and terminators in order to express the nucleicacids in a given host cell, preferably under conditions which resemblethe physiological concentrations.

[0113] The present invention further relates to a host cell containing aconstruct as defined above.

[0114] Such a host cell can be, e.g., any eukaryotic cell such as yeast,plant or mammalian, whereas human cells are preferred. Such host cellsmay form the starting material for isolation of a complex according tothe present invention.

[0115] Animal models and methods of screening for modulators (i.e.,agonists, and antagonists) of the amount of, activity of, or proteincomponent composition of, a complex of the present invention are alsoprovided.

3.1 Definitions

[0116] The term “mammalian homolog” or “homologous gene products” asused herein means a component protein of the cleavage/polyadenylationmachinery of a mammal which performs the same function as thecorresponding yeast protein. Such homologs are also termed “orthologuegene products”. The algorithm for the detection of orthologue gene pairsfrom yeast and mammalian and human uses the whole genome of theseorganisms. First, pairwise best hits are retrieved, using a fullSmith-Waterman alignment of predicted proteins. To further improvereliability, these pairs are clustered with pairwise best hits involvingDrosophila melanogaster and C. elegans proteins. Such analysis is given,e.g., in Nature, 2001, 409:860-921. The mammalian homologs of the yeastproteins according to the invention can either be isolated based on thesequence homology of the yeast genes to the mammalian genes by cloningthe respective gene applying conventional technology and expressing theprotein from such gene, or by isolating the mammalian proteins byisolating the analogous complex according to methods commonly known inthe art, and as described in Section 6, infra.

[0117] The term “protein complex machinery” as used herein means acomplex of proteins in the cell that is able to perform one or morefunctions of the wild type protein complex. The protein complex may ormay not include and/or be associated with other molecules such asnucleic acid, such as RNA or DNA, or lipids.

[0118] As used herein, the term “percent identity” means the number ofidentical residues as defined by an optimal alignment using theSmith-Waterman algorithm divided by the length of the overlap multipliedby 100. The alignment is performed by the search program (W. R. Pearson,1991, Genomics 11:635-650) with the constraint to align the maximum ofboth sequences.

[0119] As used herein, the term “derivatives” or analogs of componentproteins a or “variants” include, but are not limited, to moleculescomprising regions that are substantially homologous to the componentproteins, in various embodiments, by at least 30%, 40%, 50%, 60%, 70%,80%, 90% or 95% identity over an amino acid sequence of identical sizeor when compared to an aligned sequence in which the alignment is doneby a computer homology program known in the art, or whose encodingnucleic acid is capable of hybridizing to a sequence encoding thecomponent protein under stringent, moderately stringent, or nonstringentconditions. It means a protein which is the outcome of a modification ofthe naturally occurring protein, by amino acid substitutions, deletionsand additios, respectively, which derivatives still exhibit thebiological function of the naturally occurring protein although notnecessarily to the same degree. The biological function of such proteinscan e.g. be examined by available in vitro cleavage/polyadenylationassays as will be described below.

[0120] As used herein, the term “Therapeutics” includes, but are notlimited to, a protein complex of the present invention, the individualcomponent proteins, and analogs and derivatives (including fragments) ofthe foregoing (e.g., as described hereinabove); antibodies thereto (asdescribed hereinabove); nucleic acids encoding the component protein,and analogs or derivatives, thereof (e.g., as described hereinabove);component protein antisense nucleic acids, and agents that modulatecomplex formation and/or activity (i.e., agonists and antagonists).

[0121] “Target for therapeutic drug” means that the respective protein(target) can bind the active ingredient of a pharmaceutical compositionand thereby changes its biological activity in response to the drugbinding.

[0122] “Effector of the cleavage/polyadenylation of precursor mRNA”means a compound that is capable of binding to a member of thecleavage/polyadenylation machinery thereby altering thecleavage/polyadenylation activity of the complex. This altering can be areduction or increase in cleavage/polyadenylation activity.

[0123] The terms “polyadenylation complex”, “cleavage/polyadenylationmachinery” and “cleavage/polyadenylation complex” are usedinterchangeably herein.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] FIG. :1 shows elements of mammalian and yeast mRNA, respectively,which are involved in polyadenylation/cleavage of precursor mRNA

[0125]FIG. 2 shows a schematic representation of the gene targetingprocedure. The TAP cassette is inserted at the C-terminus of a givenyeast ORF by homologous recombination, generating the TAP-tagged fusionprotein.

[0126]FIG. 3. showns the protein pattern obtained by separation of themembers of the polyadenylation-complex of yeast using Pta1 as a baitusing TAP. Protein bands for Cft1, Cft2, Ysh1, Rna14, Pab1, Pcf11, Ref2,Pap1, Clp1, Ykl059c, Pfs2, Ygr156w, Fip1, Rna15, YKL018w, Glc7, Yth1,Ssu72, YOR179c and Pta1 (in bold) are labeled. (Further proteinsidentified as components of the yeast complex as described in theEXAMPLES-section (infra) are not stated in the figure)

[0127]FIG. 4 shows the protein pattern obtained by the separation of themembers of the polyadenylation-complex in some of the reversetagging-experiments and re-purification of a selection of the novelinteractors. The baits using TAP used for the different experiments aregiven on top of each gel picture. The band constituing the protein usedas the bait in the respective experiments is indicated by an arrow.Previously known members of the complex are listed in bold letters.(Note: only experiments using Cft1, Cft2, Pap1, Ref2, Ykl059c, Pfs2,YOR179c and Pta1 as a bait are shown and only the proteins bands ofCft1, Cft2, Ysh1, Rna14, Pab1, Ref2, Clp1, Ygr156w, Fip1, Glc7, Yht1,Yor179c, Pta1, Pcf11, Pab1, Ykl059c, Pfs2, Rna15, Ykl018w and Ssu72 arelabelled).

5. DETAILED DESCRIPTION OF THE INVENTION

[0128] The present invention relates to components of thecleavage/polyadenylation machinery of pre-cursor mRNA, the completeprotein complex, uses of said components and complex as well as tomethods of preparing same.

[0129] This is further described below. Also a description of the newlyidentified components of the cleavage/polyadenylation machinery is givenbelow.

[0130] In more detail, the present invention relates to the followingembodiments: An isolated complex selected from complex (I) andcomprising

[0131] (a) a first protein, or a functionally active fragment orfunctionally active derivative thereof, which first protein is selectedfrom the group of proteins in Table 1, column A, or a mammalian homologthereof, or a variant of said protein encoded by a nucleic acid thathybridizes to the nucleic acid of said protein or its complement underlow stringency conditions; and

[0132] (b) a second protein, or a functionally active fragment orfunctionally active derivative thereof, which second protein is selectedfrom the group of proteins in Table 1, column B, or a mammalian homologthereof, or a variant of said protein encoded by a nucleic acid thathybridizes to the nucleic acid of said protein or its complement underlow stringency conditions, wherein said first protein and said secondprotein are members of a native cellular Polyadenylation-complex, andwherein said low stringency conditions comprise hybridization in abuffer comprising 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ug/ml denatured salmonsperm DNA, and 10% (wt/vol) dextran sulfate for 18-20 hours at 40° C.,washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS for 1.5 hours at 55° C., and washing in a bufferconsisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDSfor 1.5 hours at 60° C., and a complex (II) comprising at least twosecond proteins.

[0133] The present invention further relates to a new protein complexwhich is useful for cleaving and/or polyadenylating a nucleic acid andwhich complex comprises at least one of the components according to theinvention. Such a complex can be isolated from a natural source byapplying the process according to the invention or can be reconstitutedfrom the different components made available by the present invention.

[0134] Furthermore, the invention relates to an isolated complexcomprising all proteins in column C of table 1, or the mammalianhomologs of those proteins, or variants of said proteins encoded bynucleic acid that hybridises to the nucleic acid of any of said proteinsor its complements under low stringency conditions, wherein proteins aremembers of a native cellular complex, and wherein said low stringencyconditions comprise hybridization in a buffer comprising 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2%BSA, 100 ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextransulfate for 18-20 hours at 40° C., washing in a buffer consisting of2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at55° C., and washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60° C.

[0135] Furthermore, the invention relates to an isolated complex thatcomprises all but 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18,19,20,21,22,23,24,25,26,27 or 28 of all proteins in column C oftable 1.

[0136] Furthermore, the invention relates to the complex as describedabove comprising a functionally active derivative of said first proteinand/or a functionally active derivative of said second protein, whereinthe functionally active derivative is a fusion protein comprising saidfirst protein or said second protein fused to an amino acid sequencedifferent from the first protein or second protein, respectively.

[0137] The present invention further relates to a fusion proteincomprising a component according to the invention. The fusion part,which can be added to the N-terminal, the C-terminal or into the aminoacid sequence of the component according to the invention may comprise afew amino acids only e.g. at least five, which amino acids for exampleprovide an epitope which is then be used as a target for affinitypurification of the protein and the complex, respectively. Such a typeof added amino acid is also termed “tag” throughout the presentspecification (optionally, the fusion protein may comprise even morethan one such fusion partner).

[0138] In a preferred embodiment of the present invention, the proteincomponents of the complex are vertebrate homologs of the yeast proteins,or a mixture of yeast and vertebrate homolog proteins. In a morepreferred embodiment, the protein components of the complex aremammalian homologs of the yeast proteins, or a mixture of yeast andmammalian homolog proteins. In particular aspects,n the native componentproteins, or derivatives or fragments of the complex are obtained from amammal such as mouse, rat, pig, cow, dog, monkey, human, sheep or horse.In another preferred embodiment, the protein components of the complexare human homologs of the yeast proteins, or a mixture of yeast andhuman homolog proteins. In yet another preferred embodiment, the proteincomponents of the complex are a mixture of yeast, vertebrate, mammalianand/or human proteins.

[0139] The mammalian homologs or “orthologues” of the yeast proteinsaccording to the invention can either be isolated based on the sequencehomology of the yeast genes to the mammalian genes by cloning therespective gene applying conventional technology and expressing theprotein from such gene or by isolating the mammalian proteins accordingto the process of the invention as explained in more detail below.

[0140] The derivatives of the proteins according to the invention can beproduced e.g. by recombinant DNA technology applying the standardtechnology to modify the amino acid sequence of a given protein via themodification of the underlying gene using e.g. site directedmutagenesis, etc.

[0141] A protein that shows a certain degree of identity to thenaturally occurring proteins from mammals and/or yeast, respectively,can also be prepared e.g. by applying recombinant DNA technology asdescribed above for the derivatives according to the invention.Alternatively, such protein can be isolated from natural sources byapplying the process of the invention.

[0142] Furthermore, the invention relates to a complex as describedabove that is involved in the 3′ end processing activity. Such a complexmight also exist as a module or subcomplex of a larger physiologicalprotein complex or assembly.

[0143] Furthermore, the invention relates to a complex as describedabove comprising a fragment of said first protein and/or a fragment ofsaid second protein, which fragment binds to another protein componentof said complex.

[0144] Furthermore, the invention relates to a complex as describedabove, wherein the functionally active derivative is a fusion proteincomprising said first protein or said second protein preferentiallyfused to an affinity tag or label.

[0145] It is further directed to complexes comprising a fusion proteinwhich comprises a component of the complex or a fragment thereof linkedvia a covalent bond to an amino acid sequence different from saidcomponent protein, as well as nucleic acids encoding the protein,fusions and fragments thereof. For example, the non-component proteinportion of the fusion protein, which can be added to the N-terminal, theC-terminal or inserted into the amino acid sequence of the complexcomponent can comprise a few amino acids, which provide an epitope thatis used as a target for affinity purification of the fusion proteinand/or complex.

[0146] Furthermore the invention relates to a process for processing RNAcomprising the step of bringing into contact any of the complexesdescribed above with RNA, such that RNA is processed.

[0147] Furthermore, the invention relates to an antibody or a fragmentof said antibody containing the binding domain thereof, which binds thecomplex as described above of claim and which does not bind the firstprotein when uncomplexed or the second protein when uncomplexed.

[0148] Furthermore, the invention relates to a pharmaceuticalcomposition comprising the protein complex as described above and apharmaceutically acceptable carrier.

[0149] Moreover, the present invention provides a process for theidentification and/or preparation of an effector of a compositionaccording to the invention which process comprises the steps of bringinginto contact the composition of the invention or of a component thereofwith a compound, a mixture of compounds or a library of compounds anddetermining whether the compounds or certain compounds of the mixture orlibrary bind to the composition of the invention and/or a componentthereof and/or affects the biological activity of such a composition orcomponent and then optionally further purifying the compound positivelytested as effector by such a process.

[0150] A major application of the composition according to the inventionresults in the identification of an active agent capable of bindingthereto. Hence, the compositions of the invention are useful tools inscreening for new pharmaceutical drugs.

[0151] Furthermore, the invention relates to a method for screening fora molecule that modulates directly or indirectly the function, activity,composition or formation of the complex as described above comprisingthe steps of:

[0152] (a) exposing said complex, or a cell or organism containing saidcomplex to one or more candidate molecules; and

[0153] (b) determining the amount of, the 3′ end processing activity formRNA of, or protein components of, said complex, wherein a change insaid amount, activity, or protein components relative to said amount,activity or protein components in the absence of said candidatemolecules indicates that the molecules modulate function, activity orcomposition of said complex.

[0154] Furthermore, the invention relates to a method as describedabove, wherein the amount of said complex is determined.

[0155] Furthermore, the invention relates to a method as describedabove, wherein the activity of said complex is determined.

[0156] Furthermore, the invention relates to a method as describedabove, wherein said determining step comprises isolating from the cellor organism said complex to produce said isolated complex and contactingsaid isolating complex with the substrate under conditions conducive tobinding to the complex.

[0157] Furthermore, the invention relates to a method as describedabove, wherein the protein components of said complex are determined.

[0158] Furthermore, the invention relates to a method as describedabove, wherein said determining step comprises determining whether anyof the proteins listed in column B of table 1 of said complex or themammalian homologs thereof, or variant of said proteins encoded by anucleic acid that hybridises to the nucleic acids of any of saidproteins or its complements under low stringency conditions, is presentin the complex, wherein said low stringency conditions comprisehybridization in a buffer comprising 35% formamide, 5×SSC, 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 55° C., andwashing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS for 1.5 hours at 60° C.

[0159] Furthermore, the invention relates to a method as describedabove, wherein said method is a method of screening for a drug fortreatment or prevention of diseases and disorders, preferably diseasesor disorders such as infectious diseases; viral infections such asherpes simplex infections, Epstein-Barr-infections, influenza; metabolicdisease such as metachromatic leukodystrophy; neurodegenerativedisorders such as amyotrophic lateral sclerosis and cancer.

[0160] Furthermore, the invention relates to a method for screening fora molecule that binds the complex as described above comprising thefollowing steps:

[0161] (a) exposing said complex, or a cell or organism containing saidcomplex, to one or more candidate molecules; and

[0162] (b) determining whether said complex is bound by any of saidcandidate molecules.

[0163] Furthermore, the invention relates to a method for diagnosing orscreening for the presence of a disease or disorder or a predispositionfor developing a disease or disorder in a subject, which disease ordisorder is characterized by an aberrant amount of, the 3′ endprocessing activity for mRNA biochemical activity of, or componentcomposition or formation of, the complex as described above, comprisingdetermining the amount of, the 3′ end processing activity for mRNA of,or protein components of, said complex in a sample derived from asubject, wherein a difference in said amount, activity, or proteincomponents of, said complex in an analogous sample from a subject nothaving the disease or disorder or predisposition indicates the presencein the subject of the disease or disorder or predisposition.

[0164] Furthermore, the invention relates to a method as describedabove, wherein the amount of said complex is determined.

[0165] Furthermore, the invention relates to a method as describedabove, wherein the activity of said complex is determined.

[0166] Furthermore, the invention relates to a method as describedabove, wherein said determining step comprises isolating from the cellor organism said complex to produce said isolated complex and contactingsaid isolating complex with the substrate under conditions conducive tobinding to the complex.

[0167] Furthermore, the invention relates to a method as describedabove, wherein the protein components of said complex are determined.

[0168] Furthermore, the invention relates to a method as describedabove, wherein said determining step comprises determining whether anyof the proteins listed in column B of table 1 of said complex or themammalian homologs thereof, or variant of said proteins encoded by anucleic acid that hybridises to the nucleic acids of any of saidproteins or its complements under low stringency conditions, is presentin the complex, wherein said low stringency conditions comprisehybridization in a buffer comprising 35% formamide, 5×SSC, 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 55° C., andwashing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS for 1.5 hours at 60° C.

[0169] Furthermore, the invention relates to a method for treating orpreventing a disease or disorder characterized by an aberrant amount of,the 3′ end processing activity for mRNA of, or component composition orformation of, the complex as described above, comprising administeringto a subject in need of such treatment or prevention a therapeuticallyeffective amount of one or more molecules that modulate the amount of,the 3′ end processing activity for mRNA of, or protein components orformation of, said complex.

[0170] Furthermore, the invention relates to a method as describedabove, wherein said disease or disorder involves decreased levels of theamount or activity of said complex. Furthermore, the invention relatesto a method as described above, wherein said disease or disorderinvolves increased levels of the amount or activity of said complex.

[0171] Furthermore, the invention relates to the use of a molecule thatmodulates the amount of, the 3′ end processing activity for mRNA of, orprotein components or formation of the complex as described above forthe manufacture of a medicament for the treatment or prevention of adisease or disorder, preferably diseases or disorders such as infectiousdiseases; viral infections such as herpes simplex infections,Epstein-Barr-infections, influenza; metabolic disease such asmetachromatic leukodystrophy; neurodegenerative disorders such asamyotrophic lateral sclerosis; cancer

[0172] The present invention further relates to the use of the productsaccording to the invention in therapy wherein the products according tothe invention are useful as a target for a therapeutic drug. It is knownfrom the prior art that mRNA 3′-end processing is involved in viralgrowth, in the development of cancer and in certain neurodegenerativediseases. By having identified new components of thecleavage/polyadenylation machinery the present invention, hence, offersnew targets for treating viral diseases, cancer and neurodegenerativediseases. By affecting the biological activity of the components of theinvention and/or by affecting the complex as a whole thecleavage/polyadenylation activity thereof can be influenced depending bythe needs of the patient to be treated.

[0173] The present invention further relates to a pharmaceuticalcomposition comprising a product according to the invention. Suchpharmaceutical composition contains beside the product according to theinvention as active ingredient further excipients and additives as knownby a skilled person. The present invention, hence, allows theidentification of new effectors which affect the biological activity ofthe cleavage/polyadenylation machinery of precursor RNA. Said effectorsthan can be used to modify the cleavage/polyadenylation machinery in acell by introducing an effector into a cell. Moreover, the mRNAprocessing activity of a given cell can also be affected by introducinga product according to the invention into such cell.

[0174] Furthermore, the invention relates to a kit comprising in one ormore containers

[0175] (a) an isolated first protein, or a functionally active fragmentor functionally active derivative thereof selected from the proteins incolumn A of table 1 of a given complex or a mammalian homolog thereof,or a variant of said protein encoded by a nucleic acid that hybridisesto the nucleic acid of said protein or its complement under lowstringency conditions; and

[0176] (b) an isolated second protein, or a functionally active fragmentor functionally active derivative thereof selected from the proteins incolumn B of table 1 of a given complex or a mammalian homolog thereof,or a variant of said protein encoded by a nucleic acid that hybridisesto the nucleic acid of said protein or its complement under lowstringency conditions, wherein said first and said second protein aremembers of a native cellular complex, and wherein said low stringencyconditions comprise hybridization in a buffer comprising 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2%BSA, 100 ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextransulfate for 18-20 hours at 40° C., washing in a buffer consisting of2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at55° C., and washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60° C.

[0177] Furthermore, the invention relates to a kit comprising in acontainer the isolated complex as described above or the antibody asdescribed above, optionally together with further reagents and workinginstructions. The further reagents may be, for example, buffers,substrates for enzymes but also carrier material such as beads, filters,microarrays and other solid carries. The working instructions mayindicate how to use the ingredients of the kit in order to perform adesired assay.

[0178] Furthermore, the invention relates to such kits for use inprocessing of RNA and for use in the diagnosis, prognosis and screeningin or for the diseases mentioned above.

[0179] The present invention further relates to a kit for processing RNAwhich kit comprises a product according to the invention. Such a kit maycontain e.g. expression vectors encoding the essential components of thecleavage/polyadenylation machinery which components after beingexpressed can be reconstituted in order to form a biologically activecleavage/polyadenylation complex. Such a kit preferably also containsthe required buffers and reagents together with the workinginstructions.

[0180] The present invention further relates to a kit for the diagnosisof diseases of mammals which kit comprises a product according to theinvention. As stated above the polyadenylation/cleavage machinery isinvolved in a large number of diseases. If said machinery activity ischanged due to e.g. mutations of some components thereof and/oreffectors, this may have severe implications on the affected organism.The kit according to the present invention containing the productsaccording to the invention allows to examine as to whether thecleavage/polyadenylation machinery in a given sample might show somedefects. Such a kit may be used to determine genetic defects of thegenes encoding the components of the cleavage/polyadenylation machinery.

[0181] Furthermore, the invention relates to a complex as describedabove, or the antibody or fragment as described above, for use in amethod of diagnosing a disease or disorder, preferably the diseases ordisorders as mentioned above.

[0182] Furthermore, the invention relates to a method for the productionof a pharmaceutical composition comprising carrying out the method asdescribed above to identify a molecule that modulates the function,activity or formation of said complex, and further comprising mixing theidentified molecule with a pharmaceutically acceptable carrier.

[0183] Furthermore, the invention relates to a process for preparingcomplex as described above and optionally the components thereofcomprising the following steps: expressing such a protein in a targetcell, isolating the protein complex which is attached to the taggedprotein, and optionally disassociating the protein complex and isolatingthe individual complex members.

[0184] Furthermore, the invention relates to the process as describedabove characterized in that the tagged protein comprises two differenttags which allow two separate affinity purification steps.

[0185] Furthermore, the invention relates to the process as describedabove, characterized in that two tags are separated by a cleavage sitefor a protease.

[0186] Furthermore, the invention relates to a component of the saidcomplex obtainable by a process as described above.

[0187] The present invention further relates to a composition,preferably a protein complex, which is obtainable by the methodcomprising the following steps: tagging a protein as defined above, i.e.a protein which forms part of a protein complex, with a moiety,preferably an amino acid sequence, that allows affinity purification ofthe tagged protein and expressing such protein in a target cell andisolating the protein complex which is attached to the tagged protein.The details of such purification are described in WO 00/09716 and inRigaut, G. et al. (1999), Nature Biotechnology, Vol. 17 (10): 1030-1032and further herein below. The tagging can essentially be performed withany moiety which is capable of providing a specific interaction with afurther moiety, e.g. in the sense of a ligand receptor interaction,antigen antibody interaction or the like. The tagged protein can also beexpressed in an amount in the target cell which comes close to thephysiological concentration in order to avoid a complex formation merelydue to high-concentration of the expressed protein but not reflectingthe natural occurring complex.

[0188] In a further preferred embodiment, the composition is obtained byusing a tagged protein which comprises two different tags which allowtwo different affinity purification steps. This measure allows a higherdegree of purification of the composition in question. In a furtherpreferred embodiment the tagged protein comprises two tags that areseparated by a cleavage site for a protease. This allows a step-by-steppurification on affinity columns.

[0189] Furthermore, the invention relates to a complex as describedabove and/or protein thereof as a target for an active agent of apharmaceutical, preferably a drug target in the treatment or preventionof disease or disorder, preferably diseases or disorders as mentionedabove.

[0190] Furthermore, the invention relates to the Ycl046w (SEQ ID: 59),Ygr156w (SEQ ID: 61), Yhl035c (SEQ ID:63), Ykl018w (SEQ ID:179), Ylr221c(SEQ ID: 67), Yml030w (SEQ ID:69), and Yor17c (SEQ ID:71), the mammalianhomologs/orthologs of said proteins and functionally active fragmentsand derivatives of said proteins and the mammalian homologs thereofcarrying one or more amino acid substitutions, deletions and/oradditions and the nucleic acid encoding said proteins or said homologs,orthologs and functionally active fragments and derivatives thereof.

[0191] Such a nucleic acid may be used for example to express a desiredtagged protein in a given cell for the isolation of a complex orcomponent according to the invention. Such a nucleic acid may also beused for the identification and isolation of genes from other organismsby cross species hybridization.

[0192] The component according to the invention is preferably a proteincomponent which can be further modified e.g. by carbohydrate residues.The components can either be prepared by recombinant DNA technologybased on the sequences provided by the present invention or can beisolated from a biological source by using the process according to theinvention.

[0193] The present invention further relates to a fusion proteincomprising a component according to the invention. The fusion part,which can be added to the N-terminal, the C-terminal or into the aminoacid sequence of the component according to the invention may comprise afew amino acids only e.g. at least five, which amino acids for exampleprovide an epitope which is then be used as a target for affinitypurification of the protein and the complex, respectively. Such a typeof added amino acid is also termed “tag” throughout the presentspecification (optionally, the fusion protein may comprise even morethan one such fusion partner).

[0194] The present invention further relates to a construct, preferablya vector construct, which comprises a nucleic acid as described above.Such constructs may comprise expression controlling elements such aspromoters, enhancers and terminators in order to express the nucleicacids in a given host cell, preferably under conditions which resemblethe physiological concentrations.

[0195] The present invention further relates to a construct whichcomprises the nucleic acid according to the invention and at least onefurther nucleic acid which is normally not associated with the nucleicacid according to the invention. Such a construct is preferably a vectorwhich preferably is capable of replicating in a given cell and containsthe necessary transcription control elements for expressing the nucleicacid according to the invention in a given expression system. Moreover,such vector construct may contain selection markers.

[0196] The present invention further relates to a host cell containing aconstruct as defined above.

[0197] Such a host cell can be, e.g., any eukaryotic cell such as yeast,plant or mammalian, whereas human cells are preferred. Such host cellsmay form the starting material for isolation of a complex according tothe present invention.

[0198] The present invention also relates to a host cell containing anucleic acid according to the invention or a construct according to theinvention. Such a host cell may contain an expression vector whichencodes a component according to the invention which component may serveas a bait in order to isolate the further proteins of the complex andwhich at least partly interact with the bait. Host cells can beprokaryotic and eukaryotic cells, whereas mammalian host cells arepreferred.

[0199] Animal models and methods of screening for modulators (i.e.,agonists, and antagonists) of the amount of, activity of, or proteincomponent composition of, a complex of the present invention are alsoprovided.

[0200] Below is a more detailed list of the newly identified componentsof the polyadenylation complex (see also Tab. 1). The Accession-Numberstated is the GenBank-Accession number for the protein.

[0201] Act1: Is a known and essential protein (GenBankAcc. No.BAA21512.1), which has been shown to be involved in Pol II transcriptionand has been found to be associated with histone acetylation. It servesas a structural protein.

[0202] Cka1: Is a known and non-essential protein (GenBank Acc. No.CAA86916.1), which has been found to be involved in Polymerase IIItranscription and has been found to be associated with the Casein kinaseII complex.

[0203] Eft2: The translation elongation factor EF-2 is a known proteininvolved in protein synthesis (GenBank AAB64827.1)

[0204] Eno2: Is a known and essential protein (GenBank Acc. No.AAB68019.1). It has been shown to have lyase activity and is known to beinvolved in carbohydrate metabolism.

[0205] Glc7 (YER133w) is also a known protein (GenBank Acc. No.AAC03231.1). It is also an essential protein and is a Type I proteinserine threonine phosphatase which has been implicated in distinctcellular roles, such as carbohydrate metabolism, meiosis, mitosis andcell polarity. Its occurrence in the cleavage/polyadenylation machineryhas not been known before.

[0206] Gpm1: This protein is a phosphoglycerate mutase that converts2-phosphoglyvcerate to 3-phosphoglycerate in glycolysis. It is anessential protein (GenBank: CAA81994.1)

[0207] Hhf2: Is a known and non-essential protein (GenBank Acc. No.CAA95892.1) which has been shown to be involved in DNA-binding. It haspreviously been linked to Histone octamer and the RNA polymerase Iupstream activation factor.

[0208] Hta1: Is a known and non-essential protein (GenBank Acc. No.CAA88505.1) which has DNA-binding capability and has been shown to beinvolved in polymerase II transcription.

[0209] Hsc82: Is a non-essential protein so far being associated withprotein folding. (GenBank Acc. No: CAA89919.1)

[0210] Imd2: Is an Inosine-5′-monophosphate dehydrogenase so far beingassociated with nucleotide metabolism. It is non-essential. (GenBankAcc.-No.: AAB69728.1)

[0211] Imd4: Is a non-essential protein with similiarity to Imd2 so farbeing associated with nucleotide metabolism (GenBank Acc-No.:CAA86719.1)

[0212] Met6: Is a homocysteine methyltransferase so far being associatedwith amino-acid metabolism (GenBank Acc.-No.: AAB64646.1)

[0213] Pdc1: Is a pyruvate decarboxylase isozymel so far beingassociated with carbohydrate metabolism (GenBank Acc.-No.: CAA97573.1)

[0214] Pfk1: Is a known protein (GenBank Acc. No. CAA97268.1) which haspreviously been described as part of the phosphofructokinase complex.

[0215] Ref2 (YDR195w) is a known protein (GenBank Acc. No. CAA88708.1).It is a non-essential gene product. It has been shown to be involved in3′-end formation prior to the final polyadenylation step. However, Ref2has never been identified before as a component of the 3′-end processingmachinery. Ref2 has been shown to interact with Glc7, another newcomponent of the cleavage/polyadenylation machinery.

[0216] Sec13: Is a known and essential protein (GenBank Acc. NoAAB67426.1).

[0217] Sec31: Is a known and essential protein (GenBank Acc. No.CAA98772.1)

[0218] Ssa3: Is a known and non-essential protein (GenBank Acc. No.CAA84896.1) which so far has been implicated with proteinfolding/protein transport.

[0219] Ssu72 (YNL222w) is also a known protein (GenBank Acc. No.CAA96125.1) and is an essential phylogenetically conserved protein whichhas been shown to interact with the general transcription factor TFIIB(Sua7). TFIIB is an essential component of the RNA polymerase II (RNAPII) core transcriptional machinery. It is thought that this interactionplays a role in the mechanism of start site selection by RNAP II. Thefinding according to the present invention that Ssu72 is associated withPta1 is likely to be relevant since it is believed that mRNA 3′-endformation is linked with other nuclear processes like transcription,capping and splicing. Furthermore, Ssu 72 has also been clearlyidentified in a “reverse tagging experiment” as explained herein belowby using some of the Pta1 associated proteins as bait. However, whenSsu72 itself was used as a bait associated proteins were not found mostlikely due to the fact that the addition of a C-terminal tag rendersSsu72 non-functional.

[0220] Taf60: Is a known and essential protein (GenBank Acc. No.CAA96819.1) which has been shown to be involved in Polymerase IItranscription.

[0221] Tkl1: Is a non-essential transketolase so far being associatedwith amino-acid metabolism and carbohydrate metabolism (GenBank Acc-No.:CAA89191.1)

[0222] Tsa1: Translation initiation factor elF5 which so far has been toshown to catalyze hydrolysis of GTP on the 40S ribosomalsubunit-initiation complex followed by joining to 60S ribosomal subunit.(GenBank Acc.-No.: CAA92145.1)

[0223] Tye7: Is a known protein (GenBank Acc. No. CAA99671.1). It hasbeen shown to be a basic helix-loop-helix transcription factor.

[0224] Vid24: Is a known and non-essential protein (GenBank Acc. No.CAA89320.1) which has previously been associated with proteindegradation and vesicular transport.

[0225] Vps53: Is a known protein (GenBank Acc. No. CAA89320.1) which hasbeen found to play a role in protein sorting.

[0226] YCL046w: Is a non-essential protein (GenBank Acc. No.CAA42371.1).

[0227] YGR156w is the protein product of an essential gene. This proteinalso contains a RNA binding motif. (GenBankAcc. No. CAA97170.1).

[0228] YHL035c: Is a known and non-essential protein (GenBank Acc. No.AAB65047.1). It is a member of the ATP-binding cassette superfamily.

[0229] YKL018w is also an essential protein containing a WD40 domainwhich is a typical protein binding domain. (GenBank Acc. No. CAA81853.1)

[0230] YLR221c: Is a protein of unknown function (GenBank Acc.No.AAB67410.1)

[0231] YML030w: Is a protein of unknown function (GenBank Acc. No.CAA86625.1)

[0232] YOR179c shows significant sequence similarity to Ysh1(GenBankAcc. No. CAA99388.1)

[0233] Two further proteins for which binary interactions with membersof the polyadenylation complex as known so far have been shown beforehave also been purified with the complex:

[0234] YKL059c: is the product of an essential gene and is a zincbinding protein containing a C2HC Zinc finger. The presence of thisdomain predicts a RNA binding function of YKL059c. We believe thecorresponding gene product is identical to Pfs1, a protein which hasbeen mentioned in several publications, but which has never beenannotated in the databases (for review see Keller, W. andMinvielle-Sebastia (1997). Curr Opin Cell Biol 11: 352-357). (GenBankAcc. No. CAA81896.1)

[0235] Tif4632: Is a known and non-essential protein (GenBank Acc. No.CAA96751.1) which has been shown to have an RNA-binding/translationfactor activity and is involved in protein synthesis.

[0236] Tables:

[0237] Table 1: Composition of the Complex (Cleavage/polyadenylationmachinery):

[0238] First column (‘Entry point’) lists the bait proteins (TAP-tagfusion proteins) that have been chosen for the isolation of the givencomplex. Note: in several cases, different baits have been used forvalidation in reverse tagging experiments.

[0239] Second column (‘Interactions’) briefly lists any knowninteractions between different members of the complex (Abbrevations:‘2-hybrid’: interaction as identified in yeast-2-hybrid screens;‘far-western’: interaction as identified in far-western experiments;‘coipp’: interaction as identified by co-immunoprecipitationexperiments; ‘high-throughput 2 hybrid’: interaction as identified byhigh-throughput yeast-2-hybrid screens; ‘copurification’: interaction asidentified by copurification experiments; ‘immuno-affinity-columns’:interaction as identified in experiments using immuno-affinity columns;‘in vitro binding’: interaction as identified in in-vitro-bindingexperiments. If a core complex has been known previously containingseveral of the identified proteins, the name of the complex is stated.

[0240] Third colum (‘Proteins found’) lists all proteins which have beenidentified in the particular complex.

[0241] Fourth column (‘COLUMN A, ‘Known components of the complex’)lists the components of the complex as found by Cellzome, which havebeen known to interact with other members of the complex as identifiedherein. (see also third column).

[0242] Firth column (‘COLUMN B, ‘Novel proteins’) lists the novelmembers of the complex as provided in the invention.

[0243] Sixth column (‘Column C, cleavage/polyadenylation machinery’):lists again all components of the cleavage/polyadenylation machinery asidentified herein

[0244] Seventh column (COLUMN C, ‘Activity of the complex’): List thebiochemical activities of the newly identified complex.

[0245] Eighth column (COLUMN D, ‘Proteins of unknown function’):Separately lists again the members of the newly identified complex whichpreviously have not been annotated.

[0246] Ninth column (‘localization’) indicates the localization of theidentified complex (Abbevations: c: cytoplasma; b: membrane; e:ER/Golgi/vesicles; m: mitochondria; n: nucleus; u: unknown)

[0247] Table 2: Individual Yeast Proteins of the Complexes

[0248] A) Table lists in alphabetical order all yeast proteins whichhave been identified as members of the complex presented herein.Furthermore, the SEQ ID of the proteins are listed as used herein.Further columns lists the Accession-Number of the respective sequencesin MIPS, SWISS-PROT, SGD and Genbank. In addition, where applicable, theGenBank accession numbers of the respective orthologues in humans,C.elegans and Drosophila are listed.

[0249] B) Table lists again the proteins and SEQ ID as in part A. Inaddition, the table contains an overview about what has been previouslyreported on the protein, the biochemical function thereof and thecellular function thereof as stated in YPD (Constanzo, M. C. et al.,2001, Nucl. Acid Res, 29: 75-9; Hodges, P. E. et al., 1999, Nucl. AcidsRes 27: 69-73).

[0250] Table 3: Medical Application of the Complex:

[0251] First column (‘Name of complex’) lists again the name of thecomplex as used herein.

[0252] Second column (‘Cellular role’) lists keyword on the cellularrole of the complex

[0253] Third column (‘Medical applications’) lists disorder, diseases,disease areas etc. which are treatable and/or preventable and/ordiagnosable etc. by therapeutics and methods interacting with/acting viathe complex.

[0254] Table 4: Characterization of previously undescribed individualproteins of the complexes:

[0255] The table provides data on proteins which have not been annotatedpreviously but which have now been linked to a functional complex asdescribed in table 2. Names are listed on the left. In addition thetable contains a list of motifs found by sequence analysis which hasbeen part of the invention provided herein. Futhermore, the predictedknown human orthologues are listed on the right (By SWISS-PROT Accessionnumbers). Used Abbrevations are listed at the end of the table. Thefunction of the individual proteins as deduced from the association withthe complex, the sequence analysis and the analysis of the predictedortholgues is listed in the second column (‘Putative function’).

[0256] Table 5: Overview on Experimental Steps: The tables illustratesthe construction of a yeast strain expressing a TAP-tagged bait in ahigh-throuphput fashion.

[0257] Table 6: Known and Novel Components of the yeast mRNA 3′-endprocessing machinery (the cleavage/polyadenylation complex): Top part ofthe table states the different known subcomponents of thepolyadenylation complex, the function thereof, the proteins constitutingthe different subcomplexes as known so far (including their molecularweight and sequence motifs contained in the protein). Bottom part liststhe novel components of the complex as provided herein

5.1. Protein Complexes

[0258] The protein complexes of the present invention and theircomponent proteins are described in the Tables 1,2,3,4,6 (whereas Table6 gives an overview on the construction of the yeast strains). Theprotein complexes and component proteins can be obtained by methods wellknown in the art for protein purification and recombinant proteinexpression. For example, the protein complexes of the present inventioncan be isolated using the TAP method described in Section 6, infra, andin WO 00/09716 and Rigaut et al., 1999, Nature Biotechnology17:1030-1032, which are each incorporated by reference in theirentirety. Additionally, the protein complexes can be isolated byimmunoprecipitation of the component proteins and combining theimmunoprecipitated proteins. The protein complexes can also be producedby recombinantly expressing the component proteins and combining theexpressed proteins.

[0259] The nucleic and amino acid sequences of the component proteins ofthe protein complexes of the present invention are provided herein (SEQID NOS:1-2670), and can be obtained by any method known in the art,e.g., by PCR amplification using synthetic primers hybridizable to the3′ and 5′ ends of each sequence, and/or by cloning from a cDNA orgenomic library using an oligonucleotide specific for each nucleotidesequence.

[0260] Homologs (e.g., nucleic acids encoding component proteins fromother species) or other related sequences (e.g., variants, paralogs)which are members of a native cellular protein complex can be obtainedby low, moderate or high stringency hybridization with all or a portionof the particular nucleic acid sequence as a probe, using methods wellknown in the art for nucleic acid hybridization and cloning.

[0261] Exemplary moderately stringent hybridization conditions are asfollows: prehybridization of filters containing DNA is carried out for 8hours to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl(pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/mldenatured salmon sperm DNA. Filters are hybridized for 48 hours at 65°C. in prehybridization mixture containing 100 μg/ml denatured salmonsperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Washing of filters isdone at 37° C. for 1 hour in a solution containing 2×SSC, 0.01% PVP,0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at50° C. for 45 min before autoradiography. Alternatively, exemplaryconditions of high stringency are as follows: e.g., hybridization tofilter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mMEDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M.et al., eds., 1989, Current Protocols in Molecular Biology, Vol. 1,Green Publishing Associates, Inc., and John Wiley & sons, Inc., NewYork, at p. 2.10.3). Other conditions of high stringency which may beused are well known in the art. Exemplary low stringency hybridizationconditions comprise hybridization in a buffer comprising 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2%BSA, 100 μg/ml denatured salmon sperm DNA, and 10% (wt/vol) dextransulfate for 18-20 hours at 40° C., washing in a buffer consisting of2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at55° C., and washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60° C.

[0262] For recombinant expression of one or more of the proteins, thenucleic acid containing all or a portion of the nucleotide sequenceencoding the protein can be inserted into an appropriate expressionvector, i.e., a vector that contains the necessary elements for thetranscription and translation of the inserted protein coding sequence.The necessary transcriptional and translational signals can also besupplied by the native promoter of the component protein gene, and/orflanking regions.

[0263] A variety of host-vector systems may be utilized to express theprotein coding sequence. These include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

[0264] In a preferred embodiment, a complex of the present invention isobtained by expressing the entire coding sequences of the componentproteins in the same cell, either under the control of the same promoteror separate promoters. In yet another embodiment, a derivative, fragmentor homolog of a component protein is recombinantly expressed. Preferablythe derivative, fragment or homolog of the protein forms a complex withthe other components of the complex, and more preferably forms a complexthat binds to an anti-complex antibody.

[0265] The present invention further relates to an antibody which reactswith a product according to the invention. Such an antibody might beused e.g. during purification of the machinery from a given source byaffinity purification methods. Moreover, the antibody might be used indiagnosis in order to detect changes and/or modifications of a productaccording to the invention in a given sample.

[0266] Any method available in the art can be used for the insertion ofDNA fragments into a vector to construct expression vectors containing achimeric gene consisting of appropriate transcriptional/translationalcontrol signals and protein coding sequences. These methods may includein vitro recombinant DNA and synthetic techniques and in vivorecombinant techniques (genetic recombination). Expression of nucleicacid sequences encoding a component protein, or a derivative, fragmentor homolog thereof, may be regulated by a second nucleic acid sequenceso that the gene or fragment thereof is expressed in a host transformedwith the recombinant DNA molecule(s). For example, expression of theproteins may be controlled by any promoter/enhancer known in the art. Ina specific embodiment, the promoter is not native to the gene for thecomponent protein. Promoters that may be used can be selected from amongthe many known in the art, and are chosen so as to be operative in theselected host cell.

[0267] In a specific embodiment, a vector is used that comprises apromoter operably linked to nucleic acid sequences encoding a componentprotein, or a fragment, derivative or homolog thereof, one or moreorigins of replication, and optionally, one or more selectable markers(e.g., an antibiotic resistance gene).

[0268] In another specific embodiment, an expression vector containingthe coding sequence, or a portion thereof, of a component protein,either together or separately, is made by subcloning the gene sequencesinto the EcoRI restriction site of each of the three pGEX vectors(glutathione S-transferase expression vectors; Smith and Johnson, 1988,Gene 7:31-40). This allows for the expression of products in the correctreading frame.

[0269] Expression vectors containing the sequences of interest can beidentified by three general approaches: (a) nucleic acid hybridization,(b) presence or absence of “marker” gene function, and (c) expression ofthe inserted sequences. In the first approach, coding sequences can bedetected by nucleic acid hybridization to probes comprising sequenceshomologous and complementary to the inserted sequences. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker”functions (e.g., resistance to antibiotics, occlusion body formation inbaculovirus, etc.) caused by insertion of the sequences of interest inthe vector. For example, if a component protein gene, or portionthereof, is inserted within the marker gene sequence of the vector,recombinants containing the encoded protein or portion will beidentified by the absence of the marker gene function (e.g., loss ofbeta-galactosidase activity). In the third approach, recombinantexpression vectors can be identified by assaying for the componentprotein expressed by the recombinant vector. Such assays can be based,for example, on the physical or functional properties of the interactingspecies in in vitro assay systems, e.g., formation of a complexcomprising the protein or binding to an anti-complex antibody.

[0270] Once recombinant component protein molecules are identified andthe complexes or individual proteins isolated, several methods known inthe art can be used to propagate them. Using a suitable host system andgrowth conditions, recombinant expression vectors can be propagated andamplified in quantity. As previously described, the expression vectorsor derivatives which can be used include, but are not limited to, humanor animal viruses such as vaccinia virus or adenovirus; insect virusessuch as baculovirus, yeast vectors; bacteriophage vectors such as lambdaphage; and plasmid and cosmid vectors.

[0271] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies or processes theexpressed proteins in the specific fashion desired. Expression fromcertain promoters can be elevated in the presence of certain inducers;thus expression of the genetically-engineered component proteins may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation, etc.)of proteins. Appropriate cell lines or host systems can be chosen toensure that the desired modification and processing of the foreignprotein is achieved. For example, expression in a bacterial system canbe used to produce an unglycosylated core protein, while expression inmammalian cells ensures “native” glycosylation of a heterologousprotein. Furthermore, different vector/host expression systems mayeffect processing reactions to different extents.

[0272] In other specific embodiments, a component protein or a fragment,homolog or derivative thereof, may be expressed as fusion or chimericprotein product comprising the protein, fragment, homolog, or derivativejoined via a peptide bond to a heterologous protein sequence of adifferent protein. Such chimeric products can be made by ligating theappropriate nucleic acid sequences encoding the desired amino acids toeach other by methods known in the art, in the proper coding frame, andexpressing the chimeric products in a suitable host by methods commonlyknown in the art. Alternatively, such a chimeric product can be made byprotein synthetic techniques, e.g., by use of a peptide synthesizer.Chimeric genes comprising a portion of a component protein fused to anyheterologous protein-encoding sequences may be constructed.

[0273] In particular, protein component derivatives can be made byaltering their sequences by substitutions, additions or deletions thatprovide for functionally equivalent molecules. Due to the degeneracy ofnucleotide coding sequences, other DNA sequences that encodesubstantially the same amino acid sequence as a component gene or cDNAcan be used in the practice of the present invention. These include butare not limited to nucleotide sequences comprising all or portions ofthe component protein gene that are altered by the substitution ofdifferent codons that encode a functionally equivalent amino acidresidue within the sequence, thus producing a silent change. Likewise,the derivatives of the invention include, but are not limited to, thosecontaining, as a primary amino acid sequence, all or part of the aminoacid sequence of a component protein, including altered sequences inwhich functionally equivalent amino acid residues are substituted forresidues within the sequence resulting in a silent change. For example,one or more amino acid residues within the sequence can be substitutedby another amino acid of a similar polarity that acts as a functionalequivalent, resulting in a silent alteration. Substitutes for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, the nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid.

[0274] In a specific embodiment, up to 1%, 2%, 5%, 10%, 15% or 20% ofthe total number of amino acids in the wild type protein are substitutedor deleted; or 1, 2, 3, 4, 5, or 6 amino acids are inserted, substitutedor deleted relative to the wild type protein.

[0275] In a specific embodiment of the invention, the nucleic acidsencoding a protein component and protein components consisting of orcomprising a fragment of or consisting of at least 6 (continuous) aminoacids of the protein are provided. In other embodiments, the fragmentconsists of at least 10, 20, 30, 40, or 50 amino acids of the componentprotein. In specific embodiments, such fragments are not larger than 35,100 or 200 amino acids. Derivatives or analogs of component proteinsinclude, but are not limited, to molecules comprising regions that aresubstantially homologous to the component proteins, in variousembodiments, by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%identity over an amino acid sequence of identical size or when comparedto an aligned sequence in which the alignment is done by a computerhomology program known in the art, or whose encoding nucleic acid iscapable of hybridizing to a sequence encoding the component proteinunder stringent, moderately stringent, or nonstringent conditions.

[0276] The protein component derivatives and analogs of the inventioncan be produced by various methods known in the art. The manipulationswhich result in their production can occur at the gene or protein level.For example, the cloned gene sequences can be modified by any ofnumerous strategies known in the art (Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). The sequences can be cleaved atappropriate sites with restriction endonuclease(s), followed by furtherenzymatic modification if desired, isolated, and ligated in vitro. Inthe production of the gene encoding a derivative, homolog or analog of acomponent protein, care should be taken to ensure that the modified generetains the original translational reading frame, uninterrupted bytranslational stop signals, in the gene region where the desiredactivity is encoded.

[0277] Additionally, the encoding nucleic acid sequence can be mutatedin vitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy pre-existingones, to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited to,chemical mutagenesis and in vitro site-directed mutagenesis (Hutchinsonet al., 1978, J. Biol. Chem 253:6551-6558), amplification with PCRprimers containing a mutation, etc.

[0278] Once a recombinant cell expressing a component protein, orfragment or derivative thereof, is identified, the individual geneproduct or complex can be isolated and analyzed. This is achieved byassays based on the physical and/or functional properties of the proteinor complex, including, but not limited to, radioactive labeling of theproduct followed by analysis by gel electrophoresis, immunoassay,cross-linking to marker-labeled product, etc.

[0279] The component proteins and complexes may be isolated and purifiedby standard methods known in the art (either from natural sources orrecombinant host cells expressing the complexes or proteins), includingbut not restricted to column chromatography (e.g., ion exchange,affinity, gel exclusion, reversed-phase high pressure, fast proteinliquid, etc.), differential centrifugation, differential solubility, orby any other standard technique used for the purification of proteins.Functional properties may be evaluated using any suitable assay known inthe art.

[0280] Alternatively, once a component protein or its derivative, isidentified, the amino acid sequence of the protein can be deduced fromthe nucleic acid sequence of the chimeric gene from which it wasencoded. As a result, the protein or its derivative can be synthesizedby standard chemical methods known in the art (e.g., Hunkapiller et al.,1984, Nature 310: 105-111).

[0281] Manipulations of component protein sequences may be made at theprotein level. Included within the scope of the invention is a complexin which the component proteins or derivatives and analogs that aredifferentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited to specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin, etc.

[0282] In specific embodiments, the amino acid sequences are modified toinclude a fluorescent label. In another specific embodiment, the proteinsequences are modified to have a heterofunctional reagent; suchheterofunctional reagents can be used to crosslink the members of thecomplex.

[0283] In addition, complexes of analogs and derivatives of componentproteins can be chemically synthesized. For example, a peptidecorresponding to a portion of a component protein, which comprises thedesired domain or mediates the desired activity in vitro (e.g., complexformation) can be synthesized by use of a peptide synthesizer.Furthermore, if desired, non-classical amino acids or chemical aminoacid analogs can be introduced as a substitution or addition into theprotein sequence.

[0284] In cases where natural products are suspected of being mutant orare isolated from new species, the amino acid sequence of a componentprotein isolated from the natural source, as well as those expressed invitro, or from synthesized expression vectors in vivo or in vitro, canbe determined from analysis of the DNA sequence, or alternatively, bydirect sequencing of the isolated protein. Such analysis can beperformed by manual sequencing or through use of an automated amino acidsequenator.

[0285] The complexes can also be analyzed by hydrophilicity analysis(Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824-3828). Ahydrophilicity profile can be used to identify the hydrophobic andhydrophilic regions of the proteins, and help predict their orientationin designing substrates for experimental manipulation, such as inbinding experiments, antibody synthesis, etc. Secondary structuralanalysis can also be done to identify regions of the component proteins,or their derivatives, that assume specific structures (Chou and Fasman,1974, Biochemistry 13:222-23). Manipulation, translation, secondarystructure prediction, hydrophilicity and hydrophobicity profilepredictions, open reading frame prediction and plotting, anddetermination of sequence homologies, etc., can be accomplished usingcomputer software programs available in the art.

[0286] Other methods of structural analysis including but not limited toX-ray crystallography (Engstrom, 1974 Biochem. Exp. Biol. 11:7-13), massspectroscopy and gas chromatography (Methods in Protein Science, J.Wiley and Sons, New York, 1997), and computer modeling (Fletterick andZoller, eds., 1986, Computer Graphics and Molecular Modeling, In:Current Communications in Molecular Biology, Cold Spring HarborLaboratory, Cold Spring Harbor Press, New York) can also be employed.

5.2. Antibodies to Protein Complexes

[0287] According to the present invention, a protein complex of thepresent invention comprising a first protein, or a functionally activefragment or functionally active derivative thereof, selected from thegroup consisting of proteins listed in column A of table 1; and a secondprotein, or a functionally active fragment or functionally activederivative thereof, selected from the group consisting of proteinslisted in column B of table 1, or a functionally active fragment orfunctionally active derivative thereof, can be used as an immunogen togenerate antibodies which immunospecifically bind such immunogen.According to the present invention, also a protein complex of thepresent invention can be used as an immunogen to generate antibodieswhich immunospecifically bind to such immunogen comprising all proteinslisted in column C of table 1

[0288] Such antibodies include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and an Fab expressionlibrary. In a specific embodiment, antibodies to a complex comprisinghuman protein components are produced. In another embodiment, a complexformed from a fragment of said first protein and a fragment of saidsecond protein, which fragments contain the protein domain thatinteracts with the other member of the complex, are used as an immunogenfor antibody production. In a preferred embodiment, the antibodyspecific for the complex in that the antibody does not bind theindividual protein components of the complex.

[0289] Polyclonal antibodies can be prepared as described above byimmunizing a suitable subject with a polypeptide of the invention as animmunogen. Preferred polyclonal antibody compositions are ones that havebeen selected for antibodies directed against a polypeptide orpolypeptides of the invention. Particularly preferred polyclonalantibody preparations are ones that contain only antibodies directedagainst a polypeptide or polypeptides of the invention. Particularlypreferred immunogen compositions are those that contain no other humanproteins such as, for example, immunogen compositions made using anon-human host cell for recombinant expression of a polypeptide of theinvention. In such a manner, the only human epitope or epitopesrecognized by the resulting antibody compositions raised against thisimmunogen will be present as part of a polypeptide or polypeptides ofthe invention.

[0290] The antibody titer in the immunized subject can be monitored overtime by standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized polypeptide. If desired, the antibodymolecules can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. Alternatively, antibodiesspecific for a protein or polypeptide of the invention can be selectedfor (e.g., partially purified) or purified by, e.g., affinitychromatography. For example, a recombinantly expressed and purified (orpartially purified) protein of the invention is produced as describedherein, and covalently or non-covalently coupled to a solid support suchas, for example, a chromatography column. The column can then be used toaffinity purify antibodies specific for the proteins of the inventionfrom a sample containing antibodies directed against a large number ofdifferent epitopes, thereby generating a substantially purified antibodycomposition, i.e., one that is substantially free of contaminatingantibodies. By a substantially purified antibody composition is meant,in this context, that the antibody sample contains at most only 30% (bydry weight) of contaminating antibodies directed against epitopes otherthan those on the desired protein or polypeptide of the invention, andpreferably at most 20%, yet more preferably at most 10%, and mostpreferably at most 5% (by dry weight) of the sample is contaminatingantibodies. A purified antibody composition means that at least 99% ofthe antibodies in the composition are directed against the desiredprotein or polypeptide of the invention.

[0291] At an appropriate time after immunization, e.g., when thespecific antibody titers are highest, antibody-producing cells can beobtained from the subject and used to prepare monoclonal antibodies bystandard techniques, such as the hybridoma technique originallydescribed by Kohler and Milstein, 1975, Nature 256:495-497, the human Bcell hybridoma technique (Kozbor et al., 1983, Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing hybridomas is well known (see generally CurrentProtocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons,Inc., New York, N.Y.). Hybridoma cells producing a monoclonal antibodyof the invention are detected by screening the hybridoma culturesupernatants for antibodies that bind the polypeptide of interest, e.g.,using a standard ELISA assay.

[0292] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal antibody directed against a polypeptide of theinvention can be identified and isolated by screening a recombinantcombinatorial immunoglobulin library (e.g., an antibody phage displaylibrary) with the polypeptide of interest. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO92/09690; PCT Publication No. WO 90/02809; Fuchs et al., 1991,Bio/Technology 9:1370-1372; Hay et al., 1992, Hum. Antibod. Hybridomas3:81-85; Huse et al., 1989, Science 246:1275-1281; Griffiths et al.,1993, EMBO J. 12:725-734.

[0293] Additionally, recombinant antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. A chimeric antibody is a moleculein which different portions are derived from different animal species,such as those having a variable region derived from a murine mAb and ahuman immunoglobulin constant region. (See, e.g., Cabilly et al., U.S.Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which areincorporated herein by reference in their entirety.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarily determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated hereinby reference in its entirety.) Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in PCT Publication No. WO87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al., 1988, Science 240:1041-1043; Liu etal., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J.Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al.,1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst.80:1553-1559); Morrison, 1985, Science 229:1202-1207; Oi et al., 1986,Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986,Nature 321:552-525; Verhoeyan et al., 1988, Science 239:1534; andBeidler et al., 1988, J. Immunol. 141:4053-4060.

[0294] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Such antibodies can beproduced, for example, using transgenic mice which are incapable ofexpressing endogenous immunoglobulin heavy and light chains genes, butwhich can express human heavy and light chain genes. The transgenic miceare immunized in the normal fashion with a selected antigen, e.g., allor a portion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA and IgE antibodies. For an overview of this technology for producinghuman antibodies, see Lonberg and Huszar, 1995, Int. Rev. Immunol.13:65-93). For a detailed discussion of this technology for producinghuman antibodies and human monoclonal antibodies and protocols forproducing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S. Pat.No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016; andU.S. Pat. No. 5,545,806. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

[0295] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., 1994, Bio/technology12:899-903).

[0296] Antibody fragments that contain the idiotypes of the complex canbe generated by techniques known in the art. For example, such fragmentsinclude, but are not limited to, the F(ab′)2 fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′ fragmentthat can be generated by reducing the disulfide bridges of the F(ab′)2fragment; the Fab fragment that can be generated by treating theantibody molecular with papain and a reducing agent; and Fv fragments.

[0297] In the production of antibodies, screening for the desiredantibody can be accomplished by techniques known in the art, e.g., ELISA(enzyme-linked immunosorbent assay). To select antibodies specific to aparticular domain of the complex, or a derivative thereof, one may assaygenerated hybridomas for a product that binds to the fragment of thecomplex, or a derivative thereof, that contains such a domain. Forselection of an antibody that specifically binds a complex of thepresent, or a derivative, or homolog thereof, but which does notspecifically bind to the individual proteins of the complex, or aderivative, or homolog thereof, one can select on the basis of positivebinding to the complex and a lack of binding to the individual proteincomponents.

[0298] Antibodies specific to a domain of the complex, or a derivative,or homolog thereof, are also provided.

[0299] The foregoing antibodies can be used in methods known in the artrelating to the localization and/or quantification of the complexes ofthe invention, e.g., for imaging these proteins, measuring levelsthereof in appropriate physiological samples (by immunoassay), indiagnostic methods, etc. This hold true also for a derivative, orhomolog thereof of a complex.

[0300] In another embodiment of the invention (see infra), an antibodyto a complex or a fragment of such antibodies containing the antibodybinding domain, is a Therapeutic.

5.3. Diagnostic, Prognostic, and Screening Uses of Protein Complexes

[0301] The particular protein complexes of the present invention may bemarkers of normal physiological processes, and thus have diagnosticutility. Further, definition of particular groups of patients withelevations or deficiencies of a protein complex of the presentinvention, or wherein the protein complex has a change in proteincomponent composition, can lead to new nosological classifications ofdiseases, furthering diagnostic ability.

[0302] Examples for diseases or disorders in which the complexesprovided herein are involved and/or associated with are infectiousdiseases; viral infections such as herpes simplex infections,Epstein-Barr-infections, influenza; metabolic disease such asmetachromatic leukodystrophy; neurodegenerative disorders such asamyotrophic lateral sclerosis and cancer.

[0303] Detecting levels of protein complexes, or individual componentproteins that form the complexes, or detecting levels of the mRNAsencoding the components of the complex, may be used in diagnosis,prognosis, and/or staging to follow the course of a disease state, tofollow a therapeutic response, etc.

[0304] A protein complex of the present invention and the individualcomponents of the complex and a derivative, analog or subsequencethereof, encoding nucleic acids (and sequences complementary thereto),and anti-complex antibodies and antibodies directed against individualcomponents that can form the complex, are useful in diagnostics. Theforegoing molecules can be used in assays, such as immunoassays, todetect, prognose, diagnose, or monitor various conditions, diseases, anddisorders characterized by aberrant levels of a complex or aberrantcomponent composition of a complex, or monitor the treatment of suchvarious conditions, diseases, and disorders.

[0305] In particular, such an immunoassay is carried out by a methodcomprising contacting a sample derived from a patient with ananti-complex antibody under conditions such that immunospecific bindingcan occur, and detecting or measuring the amount of any immunospecificbinding by the antibody. In a specific aspect, such binding of antibody,in tissue sections, can be used to detect aberrant complex localization,or aberrant (e.g., high, low or absent) levels of a protein complex orcomplexes. In a specific embodiment, an antibody to the complex can beused to assay a patient tissue or serum sample for the presence of thecomplex, where an aberrant level of the complex is an indication of adiseased condition. By “aberrant levels” is meant increased or decreasedlevels relative to that present, or a standard level representing thatpresent, in an analogous sample from a portion or fluid of the body, orfrom a subject not having the disorder.

[0306] The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such asWestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew known in the art.

[0307] Nucleic acids encoding the components of the protein complex andrelated nucleic acid sequences and subsequences, including complementarysequences, can be used in hybridization assays. The nucleic acidsequences, or subsequences thereof, comprising about at least 8nucleotides, can be used as hybridization probes. Hybridization assayscan be used to detect, prognose, diagnose, or monitor conditions,disorders, or disease states associated with aberrant levels of themRNAs encoding the components of a complex as described, supra. Inparticular, such a hybridization assay is carried out by a methodcomprising contacting a sample containing nucleic acid with a nucleicacid probe capable of hybridizing to component protein coding DNA orRNA, under conditions such that hybridization can occur, and detectingor measuring any resulting hybridization.

[0308] In specific embodiments, diseases and disorders involving orcharacterized by aberrant levels of a protein complex or aberrantcomplex composition can be diagnosed, or its suspected presence can bescreened for, or a predisposition to develop such disorders can bedetected, by determining the component protein composition of thecomplex, or detecting aberrant levels of a member of the complex orun-complexed component proteins or encoding nucleic acids, or functionalactivity including, but not restricted to, binding to an interactingpartner, or by detecting mutations in component protein RNA, DNA orprotein (e.g., mutations such as translocations, truncations, changes innucleotide or amino acid sequence relative to wild-type that causeincreased or decreased expression or activity of a complex, and/orcomponent protein. Such diseases and disorders include, but are notlimited to, those described in Section 5.4 and its subsections.

[0309] By way of example, levels of a protein complex and the individualcomponents of a complex can be detected by immunoassay, levels ofcomponent protein RNA or DNA can be detected by hybridization assays(e.g., Northern blots, dot blots, RNase protection assays), and bindingof component proteins to each other (e.g., complex formation) can bemeasured by binding assays commonly known in the art. Translocations andpoint mutations in component protein genes can be detected by Southernblotting, RFLP analysis, PCR using primers that preferably generate afragment spanning at least most of the gene by sequencing of genomic DNAor cDNA obtained from the patient, etc.

[0310] Assays well known in the art (e.g., assays described above suchas immunoassays, nucleic acid hybridization assays, activity assays,etc.) can be used to determine whether one or more particular proteincomplexes are present at either increased or decreased levels, or areabsent, in samples from patients suffering from a particular disease ordisorder, or having a predisposition to develop such a disease ordisorder, as compared to the levels in samples from subjects not havingsuch a disease or disorder, or having a predisposition to develop such adisease or disorder. Additionally, these assays can be used to determinewhether the ratio of the complex to the un-complexed components of thecomplex, is increased or decreased in samples from patients sufferingfrom a particular disease or disorder, or having a predisposition todevelop such a disease or disorder, as compared to the ratio in samplesfrom subjects not having such a disease or disorder. In the event thatlevels of one or more particular protein complexes (i.e., complexesformed from component protein derivatives, homologs, fragments, oranalogs) are determined to be increased in patients suffering from aparticular disease or disorder, or having a predisposition to developsuch a disease or disorder, then the particular disease or disorder, orpredisposition for a disease or disorder, can be diagnosed, haveprognosis defined for, be screened for, or be monitored by detectingincreased levels of the one or more protein complexes, increased levelsof the mRNA that encodes one or more members of the one or moreparticular protein complexes, or by detecting increased complexfunctional activity.

[0311] Accordingly, in a specific embodiment of the present invention,diseases and disorders involving increased levels of one or more proteincomplexes can be diagnosed, or their suspected presence can be screenedfor, or a predisposition to develop such disorders can be detected, bydetecting increased levels of the one or more protein complexes, themRNA encoding both members of the complex, or complex functionalactivity, or by detecting mutations in the component proteins thatstabilize or enhance complex formation, e.g., mutations such astranslocations in nucleic acids, truncations in the gene or protein,changes in nucleotide or amino acid sequence relative to wild-type, thatstabilize or enhance complex formation.

[0312] In the event that levels of one or more particular proteincomplexes are determined to be decreased in patients suffering from aparticular disease or disorder, or having a predisposition to developsuch a disease or disorder, then the particular disease or disorder orpredisposition for a disease or disorder can be diagnosed, have itsprognosis determined, be screened for, or be monitored by detectingdecreased levels of the one or more protein complexes, the mRNA thatencodes one or more members of the particular one or more proteincomplexes, or by detecting decreased protein complex functionalactivity.

[0313] Accordingly, in a specific embodiment of the invention, diseasesand disorders involving decreased levels of one or more proteincomplexes can be diagnosed, or their suspected presence can be screenedfor, or a predisposition to develop such disorders can be detected, bydetecting decreased levels of the one or more protein complexes, themRNA encoding one or more members of the one or more complexes, orcomplex functional activity, or by detecting mutations in the componentproteins that decrease complex formation, e.g., mutations such astranslocations in nucleic acids, truncations in the gene or protein,changes in nucleotide or amino acid sequence relative to wild-type, thatdecrease complex formation.

[0314] Accordingly, in a specific embodiment of the invention, diseasesand disorders involving aberrant compositions of the complexes can bediagnosed, or their suspected presence can be screened for, or apredisposition to develop such disorders can be detected, by detectingthe component proteins of one or more complexes, or the mRNA encodingthe members of the one or more complexes.

[0315] The use of detection techniques, especially those involvingantibodies against a protein complex, provides a method of detectingspecific cells that express the complex or component proteins. Usingsuch assays, specific cell types can be defined in which one or moreparticular protein complexes are expressed, and the presence of thecomplex or component proteins can be correlated with cell viability,state, health, etc.

[0316] Also embodied are methods to detect a protein complex of thepresent invention in cell culture models that express particular proteincomplexes or derivatives thereof, for the purpose of characterizing orpreparing the complexes for harvest. This embodiment includes cellsorting of prokaryotes such as but not restricted to bacteria (Davey andKell, 1996, Microbiol. Rev. 60:641-696), primary cultures and tissuespecimens from eukaryotes, including mammalian species such as human(Steele et al., 1996, Clin. Obstet. Gynecol 39:801-813), and continuouscell cultures (Orfao and Ruiz-Arguelles, 1996, Clin. Biochem. 29:5-9).Such isolations can be used as methods of diagnosis, described, supra.

5.4. Therapeutic Uses of Protein Complexes

[0317] The present invention is directed to a method for treatment orprevention of various diseases and disorders by administration of atherapeutic compound (termed herein “Therapeutic”). Such “Therapeutics”include, but are not limited to, a protein complex of the presentinvention, the individual component proteins, and analogs andderivatives (including fragments) of the foregoing (e.g., as describedhereinabove); antibodies thereto (as described hereinabove); nucleicacids encoding the component protein, and analogs or derivatives,thereof (e.g., as described hereinabove); component protein antisensenucleic acids, and agents that modulate complex formation and/oractivity (i.e., agonists and antagonists).

[0318] The protein complexes, as identified herein, are implicatedsignificantly in normal physiological processes such as RNA processingand modification.

[0319] Furthermore, the protein complexes as identified herein areimplicated in processes which are implicated in or associated withpathological conditions.

[0320] Diseases and disorders which can be treated and/or preventedand/or diagnosed by Therapeutics interacting with any of the complexesprovided herein are for example infectious diseases; viral infectionssuch as herpes simplex infections, Epstein-Barr-infections, influenza;metabolic disease such as metachromatic leukodystrophy;neurodegenerative disorders such as amyotrophic lateral sclerosis andcancer.

[0321] These disorders are treated or prevented by administration of aTherapeutic that modulates (i.e. inhibits or promotes) protein complexactivity or formation. Diseases or disorders associated with aberrantlevels of complex activity or formation, or aberrant levels or activityof the component proteins, or aberrant complex composition, may betreated by administration of a Therapeutic that modulates complexformation or activity or by the administration of a protein complex.

[0322] Therapeutic may also be administered to modulate complexformation or activity or level thereof in a microbial organism such asyeast, fungi such as candida albicans causing an infectious disease inanimals or humans.

[0323] Diseases and disorders characterized by increased (relative to asubject not suffering from the disease or disorder) complex levels oractivity can be treated with Therapeutics that antagonize (i.e., reduceor inhibit) complex formation or activity. Therapeutics that can be usedinclude, but are not limited to, the component proteins or an analog,derivative or fragment of the component protein; anti-complex antibodies(e.g., antibodies specific for the protein complex, or a fragment orderivative of the antibody containing the binding region thereof;nucleic acids encoding the component proteins; antisense nucleic acidscomplementary to nucleic acids encoding the component proteins; andnucleic acids encoding the component protein that are dysfunctional dueto, e.g., a heterologous insertion within the protein coding sequence,that are used to “knockout” endogenous protein function by homologousrecombination, see, e.g., Capecchi, 1989, Science 244:1288-1292. In oneembodiment, a Therapeutic is 1, 2 or more antisense nucleic acids whichare complementary to 1, 2, or more nucleic acids, respectfully, thatencode component proteins of a complex.

[0324] In a specific embodiment of the present invention, a nucleic acidcontaining a portion of a component protein gene in which gene sequencesflank (are both 5′ and 3′ to) a different gene sequence, is used as acomponent protein antagonist, or to promote component proteininactivation by homologous recombination (see also, Koller and Smithies,1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijistra et al., 1989,Nature 342: 435-438). Additionally, mutants or derivatives of acomponent protein that has greater affinity for another componentprotein or the complex than wild type may be administered to competewith wild type protein for binding, thereby reducing the levels ofcomplexes containing the wild type protein. Other Therapeutics thatinhibit complex function can be identified by use of known convenient invitro assays, e.g., based on their ability to inhibit complex formation,or as described in Section 5.5, infra.

[0325] In specific embodiments, Therapeutics that antagonize complexformation or activity are administered therapeutically, includingprophylactically, (1) in diseases or disorders involving an increased(relative to normal or desired) level of a complex, for example, inpatients where complexes are overactive or overexpressed; or (2) indiseases or disorders where an in vitro (or in vivo) assay (see infra)indicates the utility of antagonist administration. Increased levels ofa complex can be readily detected, e.g., by quantifying protein and/orRNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) andassaying it in vitro for RNA or protein levels, or structure and/oractivity of the expressed complex (or the encoding mRNA). Many methodsstandard in the art can be thus employed including, but not limited to,immunoassays to detect complexes and/or visualize complexes (e.g.,Western blot analysis, immunoprecipitation followed by sodium dodecylsulfate polyacrylamide gel electrophoresis [SDS-PAGE],immunocytochemistry, etc.), and/or hybridization assays to detectconcurrent expression of component protein mRNA (e.g., Northern assays,dot blot analysis, in situ hybridization, etc.).

[0326] A more specific embodiment of the present invention is directedto a method of reducing complex expression (i.e., expression of theprotein components of the complex and/or formation of the complex) bytargeting mRNAs that express the protein moieties. RNA therapeuticscurrently fall within three classes, antisense species, ribozymes, orRNA aptamers (Good et al., 1997, Gene Therapy 4:45-54).

[0327] Antisense oligonucleotides have been the most widely used. By wayof example, but not limitation, antisense oligonucleotide methodology toreduce complex formation is presented below, infra. Ribozyme therapyinvolves the administration, induced expression, etc. of small RNAmolecules with enzymatic ability to cleave, bind, or otherwiseinactivate specific RNAs, to reduce or eliminate expression ofparticular proteins (Grassi and Marini, 1996, Annals of Medicine28:499-510; Gibson, 1996, Cancer and Metastasis Reviews 15:287-299). RNAaptamers are specific RNA ligand proteins, such as for Tat and Rev RNA(Good et al., 1997, Gene Therapy 4:45-54) that can specifically inhibittheir translation. Aptamers specific for component proteins can beidentified by many methods well known in the art, for example, byaffecting the formation of a complex in the protein-protein interactionassay described, infra.

[0328] In another embodiment, the activity or levels of a componentprotein are reduced by administration of another component protein, orthe encoding nucleic acid, or an antibody that immunospecifically bindsto the component protein, or a fragment or a derivative of the antibodycontaining the binding domain thereof.

[0329] In another aspect of the invention, diseases or disordersassociated with increased levels of an component protein of the complexmay be treated or prevented by administration of a Therapeutic thatincreases complex formation if the complex formation acts to reduce orinactivate the component protein through complex formation. Suchdiseases or disorders can be treated or prevented by administration ofone component member of the complex, administration of antibodies orother molecules that stabilize the complex, etc.

[0330] Diseases and disorders associated with underexpression of acomplex, or a component protein, are treated or prevented byadministration of a Therapeutic that promotes (i.e., increases orsupplies) complex levels and/or function, or individual componentprotein function. Examples of such a Therapeutic include but are notlimited to a complex or a derivative, analog or fragment of the complexthat are functionally active (e.g., able to form a complex),un-complexed component proteins and derivatives, analogs, and fragmentsof un-complexed component proteins, and nucleic acids encoding themembers of a complex or functionally active derivatives or fragments ofthe members of the complex, e.g., for use in gene therapy. In a specificembodiment, a Therapeutic includes derivatives, homologs or fragments ofa component protein that increase and/or stabilize complex formation.Examples of other agonists can be identified using in vitro assays oranimal models, examples of which are described, infra.

[0331] In yet other specific embodiments of the present invention,Therapeutics that promote complex function are administeredtherapeutically, including prophylactically, (1) in diseases ordisorders involving an absence or decreased (relative to normal ordesired) level of a complex, for example, in patients where a complex,or the individual components necessary to form the complex, is lacking,genetically defective, biologically inactive or underactive, orunder-expressed; or (2) in diseases or disorders wherein an in vitro orin vivo assay (see, infra) indicates the utility of complex agonistadministration. The absence or decreased level of a complex, componentprotein or function can be readily detected, e.g., by obtaining apatient tissue sample (e.g., from biopsy tissue) and assaying it invitro for RNA or protein levels, structure and/or activity of theexpressed complex and/or the concurrent expression of mRNA encoding thetwo components of the complex. Many methods standard in the art can bethus employed, including but not limited to immunoassays to detectand/or visualize a complex, or the individual components of a complex(e.g., Western blot analysis, immunoprecipitation followed by sodiumdodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE],immunocytochemistry, etc.) and/or hybridization assays to detectexpression of mRNAs encoding the individual protein components of acomplex by detecting and/or visualizing component mRNA concurrently orseparately using, e.g., Northern assays, dot blot analysis, in situhybridization, etc.

[0332] In specific embodiments, the activity or levels of a componentprotein are increased by administration of another component protein ofthe same complex, or a derivative, homolog or analog thereof, a nucleicacid encoding the other component, or an agent that stabilizes orenhances the other component, or a fragment or derivative of such anagent.

[0333] Generally, administration of products of species origin orspecies reactivity (in the case of antibodies) that is the same speciesas that of the patient is preferred. Thus, in a preferred embodiment, ahuman complex, or derivative, homolog or analog thereof; nucleic acidsencoding the members of the human complex or a derivative, homolog oranalog thereof; an antibody to a human complex, or a derivative thereof;or other human agents that affect component proteins or the complex, aretherapeutically or prophylactically administered to a human patient.

[0334] Preferably, suitable in vitro or in vivo assays are utilized todetermine the effect of a specific Therapeutic and whether itsadministration is indicated for treatment of the affected tissue orindividual.

[0335] In various specific embodiments, in vitro assays can be carriedout with representative cells of cell types involved in a patient'sdisorder, to determine if a Therapeutic has a desired effect upon suchcell types.

[0336] Compounds for use in therapy can be tested in suitable animalmodel systems prior to testing in humans, including, but not limited to,rats, mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing,prior to administration to humans, any animal model system known in theart may be used. Additional descriptions and sources of Therapeuticsthat can be used according to the invention are found in Sections 5.1 to5.3 and 5.7 herein.

5.4.1. Gene Therapy

[0337] In a specific embodiment of the present invention, nucleic acidscomprising a sequence encoding the component proteins, or a functionalderivative thereof, are administered to modulate complex activity orformation by way of gene therapy. Gene therapy refers to therapyperformed by the administration of a nucleic acid to a subject. In thisembodiment of the present invention, the nucleic acid expresses itsencoded protein(s) that mediates a therapeutic effect by modulatingcomplex activity or formation. Any of the methods for gene therapyavailable in the art can be used according to the present invention.Exemplary methods are described below.

[0338] For general reviews of the methods of gene therapy, see Goldspielet al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; and May, 1993, TIBTECH 11:155-215. Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al., eds., 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

[0339] In a preferred aspect, the Therapeutic comprises a nucleic acidthat is part of an expression vector that expresses one or more of thecomponent proteins, or fragments or chimeric proteins thereof, in asuitable host. In particular, such a nucleic acid has a promoteroperably linked to the protein coding region(s) (or, less preferablyseparate promoters linked to the separate coding regions separately),said promoter being inducible or constitutive, and optionally,tissue-specific. In another particular embodiment, a nucleic acidmolecule is used in which the coding sequences, and any other desiredsequences, are flanked by regions that promote homologous recombinationat a desired site in the genome, thus providing for intra-chromosomalexpression of the component protein nucleic acids (Koller and Smithies,1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989,Nature 342:435-438).

[0340] Delivery of the nucleic acid into a patient may be either direct,in which case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

[0341] In a specific embodiment, the nucleic acid is directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing it as part of an appropriate nucleic acidexpression vector and administering it so that it becomes intracellular,e.g., by infection using a defective or attenuated retroviral or otherviral vector (U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors, orthrough use of transfecting agents, by encapsulation in liposomes,microparticles, or microcapsules, or by administering it in linkage to apeptide that is known to enter the nucleus, or by administering it inlinkage to a ligand subject to receptor-mediated endocytosis that can beused to target cell types specifically expressing the receptors (e.g.,Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), etc. In anotherembodiment, a nucleic acid-ligand complex can be formed in which theligand comprises a fusogenic viral peptide that disrupts endosomes,allowing the nucleic acid to avoid lysosomal degradation. In yet anotherembodiment, the nucleic acid can be targeted in vivo for cell specificuptake and expression, by targeting a specific receptor (see, e.g.,International Patent Publications WO 92/06180; WO 92/22635; WO 92/20316;WO 93/14188; and WO 93/20221. Alternatively, the nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller and Smithies, 1989,Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature342:435-438).

[0342] In a specific embodiment, a viral vector that contains thecomponent protein encoding nucleic acids is used. For example, aretroviral vector can be used (Miller et al., 1993, Meth. Enzymol.217:581-599). These retroviral vectors have been modified to deleteretroviral sequences that are not necessary for packaging of the viralgenome and integration into host cell DNA. The encoding nucleic acids tobe used in gene therapy is/are cloned into the vector, which facilitatesdelivery of the gene into a patient. More detail about retroviralvectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, whichdescribes the use of a retroviral vector to deliver the mdr1 gene tohematopoetic stem cells in order to make the stem cells more resistantto chemotherapy. Other references illustrating the use of retroviralvectors in gene therapy are Clowes et al., 1994, J. Clin. Invest.93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg,1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr.Opin. in Genetics and Devel. 3:110-114.

[0343] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are the liver, the central nervoussystem, endothelial cells and muscle. Adenoviruses have the advantage ofbeing capable of infecting non-dividing cells. Kozarsky and Wilson,1993, Current Opinion in Genetics and Development 3:499-503, discussadenovirus-based gene therapy. The use of adenovirus vectors to transfergenes to the respiratory epithelia of rhesus monkeys has beendemonstrated by Bout et al., 1994, Human Gene Therapy 5:3-10. Otherinstances of the use of adenoviruses in gene therapy can be found inRosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992,Cell 68:143-155; and Mastrangeli et al., 1993, J. Clin. Invest.91:225-234.

[0344] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.204:289-300.

[0345] Another approach to gene therapy involves transferring a geneinto cells in tissue culture by methods such as electroporation,lipofection, calcium phosphate-mediated transfection, or viralinfection. Usually, the method of transfer includes the transfer of aselectable marker to the cells. The cells are then placed underselection to isolate those cells that have taken up and are expressingthe transferred gene from these that have not. Those cells are thendelivered to a patient.

[0346] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the artincluding, but not limited to, transfection by electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably, is heritable and expressible by its cell progeny.

[0347] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. In a preferred embodiment, epithelialcells are injected, e.g., subcutaneously. In another embodiment,recombinant skin cells may be applied as a skin graft onto the patient.Recombinant blood cells (e.g., hematopoetic stem or progenitor cells)are preferably administered intravenously. The amount of cellsenvisioned for use depends on the desired effect, patient state, etc.,and can be determined by one skilled in the art.

[0348] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes, blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, and granulocytes, various stem orprogenitor cells, in particular hematopoetic stem or progenitor cells,e.g., as obtained from bone marrow, umbilical cord blood, peripheralblood, fetal liver, etc.

[0349] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0350] In an embodiment in which recombinant cells are used in genetherapy, a component protein encoding nucleic acid is/are introducedinto the cells such that the gene or genes are expressible by the cellsor their progeny, and the recombinant cells are then administered invivo for therapeutic effect. In a specific embodiment, stem orprogenitor cells are used. Any stem and/or progenitor cells which can beisolated and maintained in vitro can potentially be used in accordancewith this embodiment of the present invention. Such stem cells includebut are not limited to hematopoetic stem cells (HSCs), stem cells ofepithelial tissues such as the skin and the lining of the gut, embryonicheart muscle cells, liver stem cells (International Patent PublicationWO 94/08598), and neural stem cells (Stemple and Anderson, 1992, Cell71:973-985).

[0351] Epithelial stem cells (ESCs), or keratinocytes, can be obtainedfrom tissues such as the skin and the lining of the gut by knownprocedures (Rheinwald, 1980, Meth. Cell Biol. 2A:229). In stratifiedepithelial tissue such as the skin, renewal occurs by mitosis of stemcells within the germinal layer, the layer closest to the basal lamina.Similarly, stem cells within the lining of the gut provide for a rapidrenewal rate of this tissue. ESCs or keratinocytes obtained from theskin or lining of the gut of a patient or donor can be grown in tissueculture (Rheinwald, 1980, Meth. Cell Bio. 2A:229; Pittelkow and Scott,1986, Mayo Clinic Proc. 61:771). If the ESCs are provided by a donor, amethod for suppression of host versus graft reactivity (e.g.,irradiation, or drug or antibody administration to promote moderateimmunosuppression) can also be used.

[0352] With respect to hematopoetic stem cells (HSCs), any techniquethat provides for the isolation, propagation, and maintenance in vitroof HSCs can be used in this embodiment of the invention. Techniques bywhich this may be accomplished include (a) the isolation andestablishment of HSC cultures from bone marrow cells isolated from thefuture host, or a donor, or (b) the use of previously establishedlong-term HSC cultures, which may be allogeneic or xenogeneic.Non-autologous HSCs are used preferably in conjunction with a method ofsuppressing transplantation immune reactions between the future host andpatient. In a particular embodiment of the present invention, human bonemarrow cells can be obtained from the posterior iliac crest by needleaspiration (see, e.g., Kodo et al., 1984, J. Clin. Invest. 73:1377-1384). In a preferred embodiment of the present invention, the HSCscan be made highly enriched or in substantially pure form. Thisenrichment can be accomplished before, during, or after long-termculturing, and can be done by any technique known in the art. Long-termcultures of bone marrow cells can be established and maintained byusing, for example, modified Dexter cell culture techniques (Dexter etal., 1977, J. Cell Physiol. 91:335) or Witlock-Witte culture techniques(Witlock and Witte, 1982, Proc. Natl. Acad. Sci. USA 79:3608-3612).

[0353] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription.

[0354] Additional methods can be adapted for use to deliver a nucleicacid encoding the component proteins, or functional derivatives thereof,e.g., as described in Section 5.1, supra.

[0355] 5.4.2. Use of Anitsense Oligonucleotides for Suppression ofProtein Complex Activity or Formation

[0356] In a specific embodiment of the present invention, proteincomplex activity and formation is inhibited by use of antisense nucleicacids for the component proteins of the complex, that inhibittranscription and/or translation of their complementary sequence. Thepresent invention provides the therapeutic or prophylactic use ofnucleic acids of at least six nucleotides that are antisense to a geneor cDNA encoding a component protein, or a portion thereof. An“antisense” nucleic acid as used herein refers to a nucleic acid capableof hybridizing to a sequence-specific portion of a component protein RNA(preferably mRNA) by virtue of some sequence complementarity. Theantisense nucleic acid may be complementary to a coding and/or noncodingregion of a component protein mRNA. Such antisense nucleic acids thatinhibit complex formation or activity have utility as Therapeutics, andcan be used in the treatment or prevention of disorders as describedsupra.

[0357] The antisense nucleic acids of the invention can beoligonucleotides that are double-stranded or single-stranded, RNA orDNA, or a modification or derivative thereof, which can be directlyadministered to a cell, or which can be produced intracellularly bytranscription of exogenous, introduced sequences.

[0358] In another embodiment, the present invention is directed to amethod for inhibiting the expression of component protein nucleic acidsequences, in a prokaryotic or eukaryotic cell, comprising providing thecell with an effective amount of a composition comprising an antisensenucleic acid of the component protein, or a derivative thereof, of theinvention.

[0359] The antisense nucleic acids are of at least six nucleotides andare preferably oligonucleotides, ranging from 6 to about 200nucleotides. In specific aspects, the oligonucleotide is at least 10nucleotides, at least 15 nucleotides, at least 100 nucleotides, or atleast 200 nucleotides. The oligonucleotides can be DNA or RNA orchimeric mixtures, or derivatives or modified versions thereof, andeither single-stranded or double-stranded. The oligonucleotide can bemodified at the base moiety, sugar moiety, or phosphate backbone. Theoligonucleotide may include other appending groups such as peptides,agents facilitating transport across the cell membrane (see, e.g.,Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; InternationalPatent Publication No. WO 88/09810) or blood-brain barrier (see, e.g.,International Patent Publication No. WO 89/10134),hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988,BioTechniques 6:958-976), or intercalating agents (see, e.g., Zon, 1988,Pharm. Res. 5:539-549).

[0360] In a preferred aspect of the invention, an antisenseoligonucleotide is provided, preferably as single-stranded DNA. Theoligonucleotide may be modified at any position in its structure withconstituents generally known in the art.

[0361] The antisense oligonucleotides may comprise at least one modifiedbase moiety which is selected from the group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thio-uridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5N-methoxycarboxymethyluracil, 5-methoxyuracil,2-methyl-thio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0362] In another embodiment, the oligonucleotide comprises at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0363] In yet another embodiment, the oligonucleotide comprises at leastone modified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal, or an analog of the foregoing.

[0364] In yet another embodiment, the oligonucleotide is a 2-a-anomericoligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641).

[0365] The oligonucleotide may be conjugated to another molecule, e.g.,a peptide, hybridization-triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

[0366] Oligonucleotides of the invention may be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligo-nucleotides may besynthesized by the method of Stein et al. (1988, Nucl. Acids Res.16:3209), methylphosphonate oligonucleotides can be prepared by use ofcontrolled pore glass polymer supports (Sarin et al., 1988, Proc. Natl.Acad. Sci. U.S.A. 85:7448-7451), etc.

[0367] In a specific embodiment, the antisense oligonucleotides comprisecatalytic RNAs, or ribozymes (see, e.g., International PatentPublication No. WO 90/11364; Sarver et al., 1990, Science247:1222-1225). In another embodiment, the oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analog (Inoue et al., 1987, FEBSLett. 215:327-330).

[0368] In an alternative embodiment, the antisense nucleic acids of theinvention are produced intracellularly by transcription from anexogenous sequence. For example, a vector can be introduced in vivo suchthat it is taken up by a cell, within which cell the vector or a portionthereof is transcribed, producing an antisense nucleic acid (RNA) of theinvention. Such a vector would contain a sequence encoding the componentprotein. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredantisense RNA. Such vectors can be constructed by recombinant DNAtechnology methods standard in the art. Vectors can be plasmid, viral,or others known in the art to be capable of replication and expressionin mammalian cells. Expression of the sequences encoding the antisenseRNAs can be by any promoter known in the art to act in mammalian,preferably human, cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bemoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:39-42), etc.

[0369] The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a componentprotein gene, preferably a human gene. However, absolutecomplementarity, although preferred, is not required. A sequence“complementary to at least a portion of an RNA,” as referred to herein,means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids, a single strand of the duplexDNA may thus be tested, or triplex formation may be assayed. The abilityto hybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with a componentprotein RNA it may contain and still form a stable duplex (or triplex,as the case may be). One skilled in the art can ascertain a tolerable.degree of mismatch by use of standard procedures to determine themelting point of the hybridized complex.

[0370] The component protein antisense nucleic acids can be used totreat (or prevent) disorders of a cell type that expresses, orpreferably overexpresses, a protein complex.

[0371] Cell types that express or overexpress component protein RNA canbe identified by various methods known in the art. Such methods include,but are not limited to, hybridization with component protein-specificnucleic acids (e.g., by Northern blot hybridization, dot blothybridization, or in situ hybridization), or by observing the ability ofRNA from the cell type to be translated in vitro into the componentprotein by immunohistochemistry, Western blot analysis, ELISA, etc. In apreferred aspect, primary tissue from a patient can be assayed forprotein expression prior to treatment, e.g., by immunocytochemistry, insitu hybridization, or any number of methods to detect protein or mRNAexpression.

[0372] Pharmaceutical compositions of the invention (see Section 5.7,infra), comprising an effective amount of a protein component antisensenucleic acid in a pharmaceutically acceptable carrier can beadministered to a patient having a disease or disorder that is of a typethat expresses or overexpresses a protein complex of the presentinvention.

[0373] The amount of antisense nucleic acid that will be effective inthe treatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. Where possible, it is desirable to determine theantisense cytotoxicity in vitro, and then in useful animal modelsystems, prior to testing and use in humans.

[0374] In a specific embodiment, pharmaceutical compositions comprisingantisense nucleic acids are administered via liposomes, microparticles,or microcapsules. In various embodiments of the invention, it may beuseful to use such compositions to achieve sustained release of theantisense nucleic acids. In a specific embodiment, it may be desirableto utilize liposomes targeted via antibodies to specific identifiablecentral nervous system cell types (Leonetti et al., 1990, Proc. Natl.Acad. Sci. U.S.A. 87:2448-2451; Renneisen et al., 1990, J. Biol. Chem.265:16337-16342).

5.5. Assays of Protein Complexes and Derivatives and Analogs Thereof

[0375] The functional activity of a protein complex of the presentinvention, or a derivative, fragment or analog thereof, can be assayedby various methods. Potential modulators (e.g., agonists andantagonists) of complex activity or formation, e.g., anti-complexantibodies and antisense nucleic acids, can be assayed for the abilityto modulate complex activity or formation.

[0376] In one embodiment of the present invention, where one is assayingfor the ability to bind or compete with a wild-type complex for bindingto an anti-complex antibody, various immunoassays known in the art canbe used, including but not limited to competitive and non-competitiveassay systems using techniques such as radioimmunoassay, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays, immunoradiometricassays, gel diffusion precipitin reactions, immunodiffusion assays, insitu immunoassays (using colloidal gold, enzyme or radioisotope labels),western blot analysis, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays,immunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

[0377] The expression of the component protein genes (both endogenousand those expressed from cloned DNA containing the genes) can bedetected using techniques known in the art, including but not limited toSouthern hybridization (Southern, 1975, J. Mol. Biol. 98:503-517),northern hybridization (see, e.g., Freeman et al., 1983, Proc. Natl.Acad. Sci. USA 80:4094-4098), restriction endonuclease mapping (Sambrooket al., 1989, Molecular Cloning, A Laboratory Manual, 2^(nd) Ed. ColdSpring Harbor Laboratory Press, New York), RNase protection assays(Current Protocols in Molecular Biology, John Wiley and Sons, New York,1997), DNA sequence analysis, and polymerase chain reactionamplification (PCR; U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,889,818;Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. USA 85:7652-7657;Ochman et al., 1988, Genetics 120:621-623; Loh et al., 1989, Science243:217-220) followed by Southern hybridization with probes specific forthe component protein genes, in various cell types. Methods ofamplification other than PCR commonly known in the art can be employed.In one embodiment, Southern hybridization can be used to detect geneticlinkage of component protein gene mutations to physiological orpathological states. Various cell types, at various stages ofdevelopment, can be characterized for their expression of componentproteins at the same time and in the same cells. The stringency of thehybridization conditions for northern or Southern blot analysis can bemanipulated to ensure detection of nucleic acids with the desired degreeof relatedness to the specific probes used. Modifications to thesemethods and other methods commonly known in the art can be used.

[0378] Derivatives (e.g., fragments), homologs and analogs of onecomponent protein can be assayed for binding to another componentprotein in the same complex by any method known in the art, for examplethe modified yeast matrix mating test described in Section 5.6.1 infra,immunoprecipitation with an antibody that binds to the component proteincomplexed with other component proteins in the same complex, followed bysize fractionation of the immunoprecipitated proteins (e.g., bydenaturing or nondenaturing polyacrylamide gel electrophoresis), Westernblot analysis, etc.

[0379] One embodiment of the invention provides a method for screening aderivative, homolog or analog of a component protein for biologicalactivity comprising contacting said derivative, homolog or analog of thecomponent protein with the other component proteins in the same complex;and detecting the formation of a complex between said derivative,homolog or analog of the component protein and the other componentproteins; wherein detecting formation of said complex indicates thatsaid derivative, homolog or analog of has biological (e.g., binding)activity.

[0380] The invention also provides methods of modulating the activity ofa component protein that can participate in a protein complex byadministration of a binding partner of that protein or derivative,homolog or analog thereof.

[0381] In a specific embodiment of the present invention, a proteincomplex of the present invention is administered to treat or prevent adisease or disorder, since the complex and/or component proteins havebeen implicated in the disease and disorder. Accordingly, a proteincomplex or a derivative, homolog, analog or fragment thereof, nucleicacids encoding the component proteins, anti-complex antibodies, andother modulators of protein complex activity, can be tested for activityin treating or preventing a disease or disorder in in vitro and in vivoassays.

[0382] In one embodiment, a Therapeutic of the invention can be assayedfor activity in treating or preventing a disease by contacting culturedcells that exhibit an indicator of the disease in vitro, with theTherapeutic, and comparing the level of said indicator in the cellscontacted with the Therapeutic, with said level of said indicator incells not so contacted, wherein a lower level in said contacted cellsindicates that the Therapeutic has activity in treating or preventingthe disease.

[0383] In another embodiment of the invention, a Therapeutic of theinvention can be assayed for activity in treating or preventing adisease by administering the Therapeutic to a test animal that ispredisposed to develop symptoms of a disease, and measuring the changein said symptoms of the disease after administration of saidTherapeutic, wherein a reduction in the severity of the symptoms of thedisease or prevention of the symptoms of the disease indicates that theTherapeutic has activity in treating or preventing the disease. Such atest animal can be any one of a number of animal models known in the artfor disease. These animal models are well known in the art. These animalmodels include, but are not limited to those which are listed in thesection 5.6 (supra) as exemplary animald models to study any of thecomplexes provided in the invention.

5.6 Screening for Modulators of the Proetin Complexes

[0384] A complex of the present invention, the component proteins of thecomplex and nucleic acids encoding the component proteins, as well asderivatives and fragments of the amino and nucleic acids, can be used toscreen for compounds that bind to, or modulate the amount of, activityof, or protein component composition of, said complex, and thus, havepotential use as modulators, i.e., agonists or antagonists, of complexactivity, and/or complex formation, i.e., the amount of complex formed,and/or protein component composition of the complex.

[0385] Thus, the present invention is also directed to methods forscreening for molecules that bind to, or modulate the amount of,activity of, or protein component composition of, a complex of thepresent invention. In one embodiment of the invention, the method forscreening for a molecule that modulates directly or indirectly thefunction, activity or formation of a complex of the present inventioncomprises exposing said complex, or a cell or organism containing thecomplex machinery, to one or more candidate molecules under conditionsconducive to modulation; and determining the amount of, activity of, oridentities of the protein components of, said complex, wherein a changein said amount, activity, or identities relative to said amount,activity or identities in the absence of said candidate moleculesindicates that the molecules modulate function, activity or formation ofsaid complex.

[0386] In another embodiment, the present invention further relates to aprocess for the identification and/or preparation of an effector of thecleavage/polyadenylation of precursor mRNA comprising the step ofbringing into contact a product of any of claims 1 to 7 with a compound,a mixture or a library of compounds and determining whether the compoundor a certain compound of the mixture or library binds to the productand/or effects the products biological activity and optionally furtherpurifying the compound positively tested as effector.

[0387] In another embodiment, the present invention is directed to amethod for screening for a molecule that binds a protein complex of thepresent invention comprising exposing said complex, or a cell ororganism containing the complex machinery, to one or more candidatemolecules; and determining whether said complex is bound by any of saidcandidate molecules. Such screening assays can be carried out usingcell-free and cell-based methods that are commonly known in the art invitro, in vivo or ex vivo. For example, an isolated complex can beemployed, or a cell can be contacted with the candidate molecule and thecomplex can be isolated from such contacted cells and the isolatedcomplex can be assayed for activity or component composition. In anotherexample, a cell containing the complex can be contacted with thecandidate molecule and the levels of the complex in the contacted cellcan be measured. Additionally, such assays can be carried out in cellsrecombinantly expressing a component protein from column A of table 1 ofa given row, or a functionally active fragment or functionally activederivative thereof, and a component protein from column B of table 1 ofsaid row, or a functionally active fragment or functionally activederivative thereof. Additionally, such assays can also be carried out incells recombinantly expressing all component proteins from the group ofproteins in column C of table 1.

[0388] For example, assays can be carried out using recombinant cellsexpressing the protein components of a complex, to screen for moleculesthat bind to, or interfere with, or promote complex activity orformation. In preferred embodiments, polypeptide derivatives that havesuperior stabilities but retain the ability to form a complex (e.g., oneor more component proteins modified to be resistant to proteolyticdegradation in the binding assay buffers, or to be resistant tooxidative degradation), are used to screen for modulators of complexactivity or formation. Such resistant molecules can be generated, e.g.,by substitution of amino acids at proteolytic cleavage sites, the use ofchemically derivatized amino acids at proteolytic susceptible sites, andthe replacement of amino acid residues subject to oxidation, i.e.methionine and cysteine.

[0389] A particular aspect of the present invention relates toidentifying molecules that inhibit or promote formation or degradationof a complex of the present invention, e.g., using the method describedfor isolating the complex and identifying members of the complex usingthe TAP assay described in Section 6, infra, and in WO 00/09716 andRigaut et al., 1999, Nature Biotechnology 17:1030-1032, which are eachincorporated by reference in their entirety.

[0390] In another embodiment of the invention, a modulator is identifiedby administering a candidate molecule to a transgenic non-human animalexpressing the complex component proteins from promoters that are notthe native promoters of the respective proteins, more preferably wherethe candidate molecule is also recombinantly expressed in the transgenicnon-human animal. Alternatively, the method for identifying such amodulator can be carried out in vitro, preferably with a purifiedcomplex, and a purified candidate molecule.

[0391] Agents/molecules (candidate molecules) to be screened can beprovided as mixtures of a limited number of specified compounds, or ascompound libraries, peptide libraries and the like. Agents/molecules tobe screened may also include all forms of antisera, antisense nucleicacids, etc., that can modulate complex activity or formation. Exemplarycandidate molecules and libraries for screening are set forth in Section5.6.1, infra.

[0392] Screening the libraries can be accomplished by any of a varietyof commonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, 1989, Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390;Fowlkes et al., 1992, BioTechniques 13:422-427; Oldenburg et al., 1992,Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992,Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No.5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all toLadner et al.; Rebar and Pabo, 1993, Science 263:671-673; andInternational Patent Publication No. WO 94/18318.

[0393] In a specific embodiment, screening can be carried out bycontacting the library members with a complex immobilized on a solidphase, and harvesting those library members that bind to the protein (orencoding nucleic acid or derivative). Examples of such screeningmethods, termed “panning” techniques, are described by way of example inParmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992,BioTechniques 13:422-427; International Patent Publication No. WO94/18318; and in references cited hereinabove.

[0394] In a specific embodiment, fragments and/or analogs of proteincomponents of a complex, especially peptidomimetics, are screened foractivity as competitive or non-competitive inhibitors of complexformation (amount of complex or composition of complex) or activity inthe cell, which thereby inhibit complex activity or formation in thecell.

[0395] In one embodiment, agents that modulate (i.e., antagonize oragonize) complex activity or formation can be screened for using abinding inhibition assay, wherein agents are screened for their abilityto modulate formation of a complex under aqueous, or physiological,binding conditions in which complex formation occurs in the absence ofthe agent to be tested. Agents that interfere with the formation ofcomplexes of the invention are identified as antagonists of complexformation. Agents that promote the formation of complexes are identifiedas agonists of complex formation. Agents that completely block theformation of complexes are identified as inhibitors of complexformation.

[0396] Methods for screening may involve labeling the component proteinsof the complex with radioligands (e.g., ¹²⁵I or ³H), magnetic ligands(e.g., paramagnetic beads covalently attached to photobiotin acetate),fluorescent ligands (e.g., fluorescein or rhodamine), or enzyme ligands(e.g., luciferase or beta-galactosidase). The reactants that bind insolution can then be isolated by one of many techniques known in theart, including but not restricted to, co-immunoprecipitation of thelabeled complex moiety using antisera against the unlabeled bindingpartner (or labeled binding partner with a distinguishable marker fromthat used on the second labeled complex moiety), immunoaffinitychromatography, size exclusion chromatography, and gradient densitycentrifugation. In a preferred embodiment, the labeled binding partneris a small fragment or peptidomimetic that is not retained by acommercially available filter. Upon binding, the labeled species is thenunable to pass through the filter, providing for a simple assay ofcomplex formation.

[0397] Methods commonly known in the art are used to label at least oneof the component members of the complex. Suitable labeling methodsinclude, but are not limited to, radiolabeling by incorporation ofradiolabeled amino acids, e.g., ³H-leucine or ³⁵S-methionine,radiolabeling by post-translational iodination with ¹²⁵I or ¹³¹I usingthe chloramine T method, Bolton-Hunter reagents, etc., or labeling with³²P using phosphorylase and inorganic radiolabeled phosphorous, biotinlabeling with photobiotin acetate and sunlamp exposure, etc. In caseswhere one of the members of the complex is immobilized, e.g., asdescribed infra, the free species is labeled. Where neither of theinteracting species is immobilized, each can be labeled with adistinguishable marker such that isolation of both moieties can befollowed to provide for more accurate quantification, and to distinguishthe formation of homomeric from heteromeric complexes. Methods thatutilize accessory proteins that bind to one of the modified interactantsto improve the sensitivity of detection, increase the stability of thecomplex, etc., are provided.

[0398] Typical binding conditions are, for example, but not by way oflimitation, in an aqueous salt solution of 10-250 mM NaCl, 5-50 mMTris-HCl, pH 5-8, and 0.5% Triton X-100 or other detergent that improvesspecificity of interaction. Metal chelators and/or divalent cations maybe added to improve binding and/or reduce proteolysis. Reactiontemperatures may include 4, 10, 15, 22, 25, 35, or 42 degrees Celsius,and time of incubation is typically at least 15 seconds, but longertimes are preferred to allow binding equilibrium to occur. Particularcomplexes can be assayed using routine protein binding assays todetermine optimal binding conditions for reproducible binding.

[0399] The physical parameters of complex formation can be analyzed byquantification of complex formation using assay methods specific for thelabel used, e.g., liquid scintillation counting for radioactivitydetection, enzyme activity for enzyme-labeled moieties, etc. Thereaction results are then analyzed utilizing Scatchard analysis, Hillanalysis, and other methods commonly known in the arts (see, e.g.,Proteins, Structures, and Molecular Principles, 2^(nd) Edition (1993)Creighton, Ed., W.H. Freeman and Company, New York).

[0400] In a second common approach to binding assays, one of the bindingspecies is immobilized on a filter, in a microtiter plate well, in atest tube, to a chromatography matrix, etc., either covalently ornon-covalently. Proteins can be covalently immobilized using any methodwell known in the art, for example, but not limited to the method ofKadonaga and Tjian, 1986, Proc. Natl. Acad. Sci. USA 83:5889-5893, i.e.,linkage to a cyanogen-bromide derivatized substrate such asCNBr-Sepharose 4B (Pharmacia). Where needed, the use of spacers canreduce steric hindrance by the substrate. Non-covalent attachment ofproteins to a substrate include, but are not limited to, attachment of aprotein to a charged surface, binding with specific antibodies, bindingto a third unrelated interacting protein, etc.

[0401] Assays of agents (including cell extracts or a library pool) forcompetition for binding of one member of a complex (or derivativesthereof) with another member of the complex labeled by any means (e.g.,those means described above) are provided to screen for competitors orenhancers of complex formation.

[0402] In specific embodiments, blocking agents to inhibit non-specificbinding of reagents to other protein components, or absorptive losses ofreagents to plastics, immobilization matrices, etc., are included in theassay mixture. Blocking agents include, but are not restricted to bovineserum albumin, beta-casein, nonfat dried milk, Denhardt's reagent,Ficoll, polyvinylpyrolidine, nonionic detergents (NP40, Triton X-100,Tween 20, Tween 80, etc.), ionic detergents (e.g., SDS, LDS, etc.),polyethylene glycol, etc. Appropriate blocking agent concentrationsallow complex formation.

[0403] After binding is performed, unbound, labeled protein is removedin the supernatant, and the immobilized protein retaining any bound,labeled protein is washed extensively. The amount of bound label is thenquantified using standard methods in the art to detect the label asdescribed, supra.

[0404] Moreover, a number of polyadenylation assays are described in theprior art. Such assays can be found in Bienroth, S. E.; Wahle, C.;Suter-Crazzolara, C. and Keller, W. (1991), J. Biol. Chem. 266,19768-19776; Edwards-Gilbert, G. and Milcarek, C. (1995), Mol. Cell.Biol. 15, 6420-6429; Wahle, E. (1991), Cell 66, 759-768; Christofori, G.and Keller, W. (Cell) 54, 875-889.

[0405] Exemplary assays useful to measure the 3′ end processing activityfor mRNA of complex 162 include, but are not limited to those describedin Kessler M M et al, 1996, J Biol. Chem. 271: 27167-75, and Butler, S.J. and Platt, T. (1988), Science 242, 1270-1274, and Moore, C. L. andSharp, P. A. (1985), Cell 41, 845-855

[0406] Exemplary assays useful to measure the cleavage step in 3′ endprocessing activity of mRNA of complex 162 include, but are not limitedto those described in Ruegsegger U et al., 1996, J Biol Chem 271:6107-6113.

[0407] An exemplary RNA binding assay can be carried out by contacting acomplex having RNA binding activity with a radioactive [32P] end-labeledRNA substrate, e.g. a poly (A) RNA, under appropriate conditions anddetecting bound protein. The detection of bound protein can be carriedout, e.g., by filtrating the solution through a nitrocellulose filterand determining the radioactivity bound to the filter. This assay isbased on the retention of nucleic acid-protein complexes onNitrocellulose whereas free nucleic acid can pass through the filter

[0408] (see e.g. Wahle, E., 1991, Methods 66: 759-68)

[0409] An exemplary RNA exonuclease assay can be carried out bycontacting a complex having RNA exonuclease activity with aradioactivity [32 phosphate] end-labeled RNA substrate under appropriateconditions and detecting the release of free radioactive nucleotides.The detection of free radioactive nucleotides can be carried out, e.g.,by adding 20% trichloroacetic acid, filtrating the solution through afilter and measuring the amount of acid-soluble radioactivity

[0410] (see e.g. Ross, J., 1999, Methods 17: 52-9)

[0411] An exemplary mRNA splicing assay can be carried out by contactinga complex having mRNA splicing activity with a radioactively labeled RNAsubstrate under appropriate conditions and detecting the release ofspliced RNA species. The detection of spliced RNA species can be carriedout, e.g., by fractionation of processed RNAs in a glycerol gradient andsubsequent analysis by denaturing polyacrylamide gel elecrophoresis andvisualization by autoradiography.

[0412] (see e.g. Schwer, B. and Gross, C H., 1998, Methods17: 2086-94)

[0413] An exemplary rRNA processing assay can be carried out bycontacting a complex having rRNA processing activity with an pre-rRNAsubstrate under appropriate conditions and detecting the release of freeprocessed rRNA species. The detection of processed rRNA species can becarried out, e.g., using a primer extension or northern blotting assayby measuring the size of the rRNA species.

[0414] (see e.g. Kressler, D. et al, 1997, Methods 17: 7283-94)

[0415] 5.6.1. Candidate Molecules

[0416] Any molecule known in the art can be tested for its ability tomodulate (increase or decrease) the amount of, activity of, or proteincomponent composition of a complex of the present invention as detectedby a change in the amount of, activity of, or protein componentcomposition of, said complex. By way of example, a change in the amountof the complex can be detected by detecting a change in the amount ofthe complex that can be isolated from a cell expressing the complexmachinery. For identifying a molecule that modulates complex activity,candidate molecules can be directly provided to a cell expressing thecomplex machinery, or, in the case of candidate proteins, can beprovided by providing their encoding nucleic acids under conditions inwhich the nucleic acids are recombinantly expressed to produce thecandidate proteins within the cell expressing the complex machinery, thecomplex is then isolated from the cell and the isolated complex isassayed for activity using methods well known in the art, not limited tothose described, supra.

[0417] This embodiment of the invention is well suited to screenchemical libraries for molecules which modulate, e.g., inhibit,antagonize, or agonize, the amount of, activity of, or protein componentcomposition of the complex. The chemical libraries can be peptidelibraries, peptidomimetic libraries, chemically synthesized libraries,recombinant, e.g., phage display libraries, and in vitrotranslation-based libraries, other non-peptide synthetic organiclibraries, etc.

[0418] Exemplary libraries are commercially available from severalsources (ArQule, Tripos/PanLabs, ChemDesign, Pharmacopoeia). In somecases, these chemical libraries are generated using combinatorialstrategies that encode the identity of each member of the library on asubstrate to which the member compound is attached, thus allowing directand immediate identification of a molecule that is an effectivemodulator. Thus, in many combinatorial approaches, the position on aplate of a compound specifies that compound's composition. Also, in oneexample, a single plate position may have from 1-20 chemicals that canbe screened by administration to a well containing the interactions ofinterest. Thus, if modulation is detected, smaller and smaller pools ofinteracting pairs can be assayed for the modulation activity. By suchmethods, many candidate molecules can be screened.

[0419] Many diversity libraries suitable for use are known in the artand can be used to provide compounds to be tested according to thepresent invention. Alternatively, libraries can be constructed usingstandard methods. Chemical (synthetic) libraries, recombinant expressionlibraries, or polysome-based libraries are exemplary types of librariesthat can be used.

[0420] The libraries can be constrained or semirigid (having some degreeof structural rigidity), or linear or nonconstrained. The library can bea cDNA or genomic expression library, random peptide expression libraryor a chemically synthesized random peptide library, or non-peptidelibrary. Expression libraries are introduced into the cells in which theassay occurs, where the nucleic acids of the library are expressed toproduce their encoded proteins.

[0421] In one embodiment, peptide libraries that can be used in thepresent invention may be libraries that are chemically synthesized invitro. Examples of such libraries are given in Houghten et al., 1991,Nature 354:84-86, which describes mixtures of free hexapeptides in whichthe first and second residues in each peptide were individually andspecifically defined; Lam et al., 1991, Nature 354:82-84, whichdescribes a “one bead, one peptide” approach in which a solid phasesplit synthesis scheme produced a library of peptides in which each beadin the collection had immobilized thereon a single, random sequence ofamino acid residues; Medynski, 1994, Bio/Technology 12:709-710, whichdescribes split synthesis and T-bag synthesis methods; and Gallop etal., 1994, J. Medicinal Chemistry 37(9):1233-1251. Simply by way ofother examples, a combinatorial library may be prepared for use,according to the methods of Ohlmeyer et al., 1993, Proc. Natl. Acad.Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA91:11422-11426; Houghten et al., 1992, Biotechniques 13:412;Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; orSalmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712. PCTPublication No. WO 93/20242 and Brenner and Lerner, 1992, Proc. Natl.Acad. Sci. USA 89:5381-5383 describe “encoded combinatorial chemicallibraries,” that contain oligonucleotide identifiers for each chemicalpolymer library member.

[0422] In a preferred embodiment, the library screened is a biologicalexpression library that is a random peptide phage display library, wherethe random peptides are constrained (e.g., by virtue of having disulfidebonding).

[0423] Further, more general, structurally constrained, organicdiversity (e.g., nonpeptide) libraries, can also be used. By way ofexample, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc.Natl. Acad. Sci. USA 91:4708-4712) may be used.

[0424] Conformationally constrained libraries that can be used includebut are not limited to those containing invariant cysteine residueswhich, in an oxidizing environment, cross-link by disulfide bonds toform cystines, modified peptides (e.g., incorporating fluorine, metals,isotopic labels, are phosphorylated, etc.), peptides containing one ormore non-naturally occurring amino acids, non-peptide structures, andpeptides containing a significant fraction of -carboxyglutamic acid.

[0425] Libraries of non-peptides, e.g., peptide derivatives (forexample, that contain one or more non-naturally occurring amino acids)can also be used. One example of these are peptoid libraries (Simon etal., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371). Peptoids arepolymers of non-natural amino acids that have naturally occurring sidechains attached not to the alpha carbon but to the backbone aminonitrogen. Since peptoids are not easily degraded by human digestiveenzymes, they are advantageously more easily adaptable to drug use.Another example of a library that can be used, in which the amidefunctionalities in peptides have been permethylated to generate achemically transformed combinatorial library, is described by Ostresh etal., 1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).

[0426] The members of the peptide libraries that can be screenedaccording to the invention are not limited to containing the 20naturally occurring amino acids. In particular, chemically synthesizedlibraries and polysome based libraries allow the use of amino acids inaddition to the 20 naturally occurring amino acids (by their inclusionin the precursor pool of amino acids used in library production). Inspecific embodiments, the library members contain one or morenon-natural or non-classical amino acids or cyclic peptides.Non-classical amino acids include but are not limited to the D-isomersof the common amino acids, -amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid; -Abu, -Ahx, 6-amino hexanoic acid; Aib,2-amino isobutyric acid; 3-amino propionic acid; omithine; norleucine;norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, designer amino acids such as β-methyl amino acids, C-methylamino acids, N-methyl amino acids, fluoro-amino acids and amino acidanalogs in general. Furthermore, the amino acid can be D (dextrorotary)or L (levorotary).

[0427] In a specific embodiment, fragments and/or analogs of complexesof the invention, or protein components thereof, especiallypeptidomimetics, are screened for activity as competitive ornon-competitive inhibitors of complex activity or formation.

[0428] In another embodiment of the present invention, combinatorialchemistry can be used to identify modulators of a the complexes.Combinatorial chemistry is capable of creating libraries containinghundreds of thousands of compounds, many of which may be structurallysimilar. While high throughput screening programs are capable ofscreening these vast libraries for affinity for known targets, newapproaches have been developed that achieve libraries of smallerdimension but which provide maximum chemical diversity. (See e.g.,Matter, 1997, Journal of Medicinal Chemistry 40:1219-1229).

[0429] One method of combinatorial chemistry, affinity fingerprinting,has previously been used to test a discrete library of small moleculesfor binding affinities for a defined panel of proteins. The fingerprintsobtained by the screen are used to predict the affinity of theindividual library members for other proteins or receptors of interest(in the instant invention, the protein complexes of the presentinvention and protein components thereof.) The fingerprints are comparedwith fingerprints obtained from other compounds known to react with theprotein of interest to predict whether the library compound mightsimilarly react. For example, rather than testing every ligand in alarge library for interaction with a complex or protein component, onlythose ligands having a fingerprint similar to other compounds known tohave that activity could be tested. (See, e.g., Kauvar et al., 1995,Chemistry and Biology 2:107-118; Kauvar, 1995, Affinity fingerprinting,Pharmaceutical Manufacturing International. 8:25-28; and Kauvar,Toxic-Chemical Detection by Pattern Recognition in New Frontiers inAgrochemical Immunoassay, D. Kurtz. L. Stanker and J. H. Skerritt.Editors, 1995, AOAC: Washington, D.C., 305-312).

[0430] Kay et al., 1993, Gene 128:59-65 (Kay) discloses a method ofconstructing peptide libraries that encode peptides of totally randomsequence that are longer than those of any prior conventional libraries.The libraries disclosed in Kay encode totally synthetic random peptidesof greater than about 20 amino acids in length. Such libraries can beadvantageously screened to identify complex modulators. (See also U.S.Pat. No. 5,498,538 dated Mar. 12, 1996; and PCT Publication No. WO94/18318 dated Aug. 18, 1994).

[0431] A comprehensive review of various types of peptide libraries canbe found in Gallop et al., 1994, J. Med. Chem. 37:1233-1251.

5.7. Pharmaceutical Compositions and Therapeutic/ProphylacticAdministration

[0432] The invention provides methods of treatment (and prophylaxis) byadministration to a subject of an effective amount of a Therapeutic ofthe invention. In a preferred aspect, the Therapeutic is substantiallypurified. The subject is preferably an animal including, but not limitedto animals such as cows, pigs, horses, chickens, cats, dogs, etc., andis preferably a mammal, and most preferably human. In a specificembodiment, a non-human mammal is the subject.

[0433] Various delivery systems are known and can be used to administera Therapeutic of the invention, e.g., encapsulation in liposomes,microparticles, and microcapsules: use of recombinant cells capable ofexpressing the Therapeutic, use of receptor-mediated endocytosis (e.g.,Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432); construction of aTherapeutic nucleic acid as part of a retroviral or other vector, etc.Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compounds may be administered by anyconvenient route, for example by infusion, by bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oral,rectal and intestinal mucosa, etc.), and may be administered togetherwith other biologically active agents. Administration can be systemic orlocal. In addition, it may be desirable to introduce the pharmaceuticalcompositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0434] In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment. This may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

[0435] In another embodiment, the Therapeutic can be delivered in avesicle, in particular a liposome (Langer, 1990, Science 249:1527-1533;Treat et al., 1989, In: Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler, eds., Liss, New York, pp.353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

[0436] In yet another embodiment, the Therapeutic can be delivered via acontrolled release system. In one embodiment, a pump may be used(Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201-240;Buchwald et al., 1980, Surgery 88:507-516; Saudek et al., 1989, N. Engl.J. Med. 321:574-579). In another embodiment, polymeric materials can beused (Medical Applications of Controlled Release, Langer and Wise, eds.,CRC Press, Boca Raton, Fla., 1974; Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball, eds., Wiley, New York,1984; Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem.23:61; Levy et al., 1985, Science 228:190-192; During et al., 1989, Ann.Neurol. 25:351-356; Howard et al., 1989, J. Neurosurg. 71:858-863). Inyet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (e.g., Goodson, 1984, In: MedicalApplications of Controlled Release, supra, Vol. 2, pp. 115-138). Othercontrolled release systems are discussed in the review by Langer (1990,Science 249:1527-1533).

[0437] In a specific embodiment where the Therapeutic is a nucleic acidencoding a protein Therapeutic, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or bycoating it with lipids, cell-surface receptors or transfecting agents,or by administering it in linkage to a homeobox-like peptide which isknown to enter the nucleus (e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid Therapeuticcan be introduced intracellularly and incorporated by homologousrecombination within host cell DNA for expression.

[0438] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of aTherapeutic, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly, in humans. The term “carrier” refers toa diluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, including but not limited to peanut oil, soybean oil,mineral oil, sesame oil and the like. Water is a preferred carrier whenthe pharmaceutical composition is administered orally. Saline andaqueous dextrose are preferred carriers when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions are preferably employed as liquidcarriers for injectable solutions. Suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsions,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of theTherapeutic, preferably in purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. The formulation should suit the mode of administration.

[0439] In a preferred embodiment, the composition is formulated, inaccordance with routine procedures, as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lidocaine toease pain at the site of the injection. Generally, the ingredients aresupplied either separately or mixed together in unit dosage form, forexample, as a dry lyophilized powder or water-free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile water orsaline for injection can be provided so that the ingredients may bemixed prior to administration.

[0440] The Therapeutics of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withfree carboxyl groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., those formed with freeamine groups such as those derived from isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc., and those derived fromsodium, potassium, ammonium, calcium, and ferric hydroxides, etc.

[0441] The amount of the Therapeutic of the invention which will beeffective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20-500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

[0442] Suppositories generally contain active ingredient in the range of0.5% to 10% by weight; oral formulations preferably contain 10% to 95%active ingredient.

[0443] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

[0444] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention. Forexample, the kit can comprise in one or more containers a first protein,or a functionally active fragment or functionally active derivativethereof, which first protein is selected from the group consisting ofproteins listed in column A of table 1 of a given row; and a secondprotein, or a functionally active fragment or functionally activederivative thereof, which second protein is selected from the groupconsisting of proteins listed in column B of table 1 of said row.Alternatively, the kit can comprise in one or more containers, allproteins, functionally active fragments or functionally activederivatives thereof of from the group of proteins in column C of table1.

[0445] The kits of the present invention can also contain expressionvectors encoding the essential components of the complex machinery,which components after being expressed can be reconstituted in order toform a biologically active complex. Such a kit preferably also containsthe required buffers and reagents. Optionally associated with suchcontainer(s) can be instructions for use of the kit and/or a notice inthe form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, which noticereflects approval by the agency of manufacture, use or sale for humanadministration.

5.8 Animal Models

[0446] The present invention also provides animal models. In oneembodiment, animal models for diseases and disorders involving theprotein complexes of the present invention are provided. These animalmodels are well known in the art. These animal models include, but arenot limited to those which are listed in the section 5.6 (supra) asexemplary animaid models to study any of the complexes provided in theinvention. Such animals can be initially produced by promotinghomologous recombination or insertional mutagenesis between genesencoding the protein components of the complexes in the chromosome, andexogenous genes encoding the protein components of the complexes thathave been rendered biologically inactive or deleted (preferably byinsertion of a heterologous sequence, e.g., an antibiotic resistancegene). In a preferred aspect, homologous recombination is carried out bytransforming embryo-derived stem (ES) cells with one or more vectorscontaining one or more insertionally inactivated genes, such thathomologous recombination occurs, followed by injecting the transformedES cells into a blastocyst, and implanting the blastocyst into a fostermother, followed by the birth of the chimeric animal (“knockout animal”)in which a gene encoding a component protein from column A of table 1 ofa given row, or a functionally active fragment or functionally activederivative thereof, and a gene encoding a component protein from columnB of table 1 of said row, or a functionally active fragment orfunctionally active derivative thereof, has been inactivated or deleted(Capecchi, 1989, Science 244:1288-1292).

[0447] In another preferred aspect, homologous recombination is carriedout by transforming embryo-derived stem (ES) cells with one or morevectors containing one or more insertionally inactivated genes, suchthat homologous recombination occurs, followed by injecting thetransformed ES cells into a blastocyst, and implanting the blastocystinto a foster mother, followed by the birth of the chimeric animal(“knockout animal”) in which the genes of all component proteins fromthe group of proteins listed in column C of table 1 or of all proteinsfrom the group of proteins listed in columb D of table 1 have beeninactivated or deleted.

[0448] The chimeric animal can be bred to produce additional knockoutanimals. Such animals can be mice, hamsters, sheep, pigs, cattle, etc.,and are preferably non-human mammals. In a specific embodiment, aknockout mouse is produced.

[0449] Such knockout animals are expected to develop, or be predisposedto developing, diseases or disorders associated with mutations involvingthe protein complexes of the present invention, and thus, can have useas animal models of such diseases and disorders, e.g., to screen for ortest molecules (e.g., potential Therapeutics) for such diseases anddisorders.

[0450] In a different embodiment of the invention, transgenic animalsthat have incorporated and express (or over-express or mis-express) afunctional gene encoding a protein component of the complex, e.g. byintroducing the a gene encoding one or more of the components of thecomplex under the control of a heterologous promoter (i.e., a promoterthat is not the native promoter of the gene) that either over-expressesthe protein or proteins, or expresses them in tissues not normallyexpressing the complexes or proteins, can have use as animal models ofdiseases and disorders characterized by elevated levels of the proteincomplexes. Such animals can be used to screen or test molecules for theability to treat or prevent the diseases and disorders cited supra.

[0451] In one embodiment, the present invention provides a recombinantnon-human animal in which an endogenous gene encoding a first protein,or a functionally active fragment or functionally active derivativethereof, which first protein is selected from the group of proteins ofcolumn A of table 2 of a given complex, and and endogenous gene encodinga second protein, or a functionally active fragment or functionallyactive derivative thereof, which second protein is selected from thegroup consisting of proteins of column B, of table 2 of said complex hasbeen deleted or inactivated by homologous recombination or insertionalmutagenesis of said animal or an ancestor thereof. In addition, thepresent invention provides a recombinant non-human animal in which theendogenous genes of all proteins, or functionally active fragments orfunctionally active derivatives thereof of one of the group of proteinslisted in column C have been deleted or inactivated by homologousrecombination or insertional mutagenesis of said animal or an ancestorthereof:

[0452] In another embodiment, the present invention provides arecombinant non-human animal in which an endogenous gene encoding afirst protein, or a functionally active fragment or functionally activederivative thereof, which first protein is selected from the groupconsisting of proteins of column A of table 2 of a given complex, andendogenous gene encoding a second protein, or a functionally activefragment or functionally active derivative thereof, which second proteinis selected from the group consisting of proteins of column B, of table2 of said complex are recombinantly expressed in said animal or anancestor thereof.

[0453] The following series of examples are presented by way ofillustration and not by way of limitation on the scope of the invention.

EXAMPLES

[0454] By applying the process according to the invention to theisolation of the polyadenylation/cleavage machinery from yeast, which isfurther described below, thirty-two new proteins could be identified insaid yeast complex.

[0455] Purifications have been done using different proteins as baitaccording to the protocols stated further below.

[0456] Below is a more detailed list of the newly identified componentsof the polyadenylation complex (see also Tab. 1). The Accession-Numberstated is the GenBank-Accession number for the protein.

[0457] Protein patterns for some of the purifications are shown in FIGS.3 and 4.

[0458] Act1: Is a known and essential protein (GenBank Acc. No.BAA21512.1), which has been shown to be involved in Pol II transcriptionand has been found to be associated with histone acetylation. It servesas a structural protein.

[0459] Cka1: Is a known and non-essential protein (GenBank Acc. No.CAA86916.1), which has been found to be involved in Polymerase IIItranscription and has been found to be associated with the Casein kinaseII complex.

[0460] Eft2: The translation elongation factor EF-2 is a known proteininvolved in protein synthesis (GenBank AAB64827.1)

[0461] Eno2: Is a known and essential protein (GenBank Acc. No.AAB68019.1). It has been shown to have lyase activity and is known to beinvolved in carbohydrate metabolism.

[0462] Glc7 (YER1 33w) is also a known protein (GenBank Acc. No.AAC03231.1). It is also an essential protein and is a Type I proteinserine threonine phosphatase which has been implicated in distinctcellular roles, such as carbohydrate metabolism, meiosis, mitosis andcell polarity. Its occurrence in the cleavage/polyadenylation machineryhas not been known before.

[0463] Gpm1: This protein is a phosphoglycerate mutase that converts2-phosphoglyvcerate to 3-phosphoglycerate in glycolysis. It is anessential protein (GenBank: CAA81994.1)

[0464] Hhf2: Is a known and non-essential protein (GenBank Acc. No.CAA95892.1) which has been shown to be involved in DNA-binding. It haspreviously been linked to Histone octamer and the RNA polymerase Iupstream activation factor.

[0465] Hta1: Is a known and non-essential protein (GenBank Acc. No.CAA88505.1) which has DNA-binding capability and has been shown to beinvolved in polymerase II transcription.

[0466] Hsc82: Is a non-essential protein so far being associated withprotein folding. (GenBank Acc. No: CAA89919.1)

[0467] Imd2: Is an Inosine-5′-monophosphate dehydrogenase so far beingassociated with nucleotide metabolism. It is non-essential. (GenBankAcc.-No.: AAB69728.1)

[0468] Imd4: Is a non-essential protein with similiarity to Imd2 so farbeing associated with nucleotide metabolism (GenBank Acc-No.:CAA86719.1)

[0469] Met6: Is a homocysteine methyltransferase so far being associatedwith amino-acid metabolism (GenBank Acc.-No.: AAB64646.1)

[0470] Pdc1: Is a pyruvate decarboxylase isozymel so far beingassociated with carbohydrate metabolism (GenBank Acc.-No.: CAA97573.1)

[0471] Pfk1: Is a known protein (GenBank Acc. No. CAA97268.1) which haspreviously been described as part of the phosphofructokinase complex.

[0472] Ref2 (YDR195w) is a known protein (GenBank Acc. No. CAA88708.1).It is a non-essential gene product. It has been shown to be involved in3′-end formation prior to the final polyadenylation step. However, Ref2has never been identified before as a component of the 3′-end processingmachinery. Ref2 has been shown to interact with Glc7, another newcomponent of the cleavage/polyadenylation machinery.

[0473] Sec13: Is a known and essential protein (GenBank Acc. NoAAB67426.1).

[0474] Sec31: Is a known and essential protein (GenBank Acc. No.CAA98772.1)

[0475] Ssa3: Is a known and non-essential protein (GenBank Acc. No.CAA84896.1) which so far has been implicated with proteinfolding/protein transport.

[0476] Ssu72 (YNL222w) is also a known protein (GenBank Acc. No.CAA96125.1) and is an essential phylogenetically conserved protein whichhas been shown to interact with the general transcription factor TFIIB(Sua7). TFIIB is an essential component of the RNA polymerase II (RNAPII) core transcriptional machinery. It is thought that this interactionplays a role in the mechanism of start site selection by RNAP II. Thefinding according to the present invention that Ssu72 is associated withPta1 is likely to be relevant since it is believed that mRNA 3′-endformation is linked with other nuclear processes like transcription,capping and splicing. Furthermore, Ssu 72 has also been clearlyidentified in a “reverse tagging experiment” as explained herein belowby using some of the Pta1 associated proteins as bait. However, whenSsu72 itself was used as a bait associated proteins were not found mostlikely due to the fact that the addition of a C-terminal tag rendersSsu72 non-functional.

[0477] Taf60: Is a known and essential protein (GenBank Acc. No.CAA96819.1) which has been shown to be involved in Polymerase IItranscription.

[0478] Tkl1: Is a non-essential transketolase so far being associatedwith amino-acid metabolism and carbohydrate metabolism (GenBank Acc-No.:CAA89191.1)

[0479] Tsa1: Translation initiation factor eIF5 which so far has been toshown to catalyze hydrolysis of GTP on the 40S ribosomalsubunit-initiation complex followed by joining to 60S ribosomal subunit.(GenBankAcc.-No.: CAA92145.1)

[0480] Tye7: Is a known protein (GenBank Acc. No. CAA99671.1). It hasbeen shown to be a basic helix-loop-helix transcription factor.

[0481] Vid24: Is a known and non-essential protein (GenBank Acc. No.CAA89320.1) which has previously been associated with proteindegradation and vesicular transport.

[0482] Vps53: Is a known protein (GenBank Acc. No. CAA89320.1) which hasbeen found to play a role in protein sorting.

[0483] YCL046w: Is a non-essential protein (GenBank Acc. No.CAA42371.1).

[0484] YGR156w is the protein product of an essential gene. This proteinalso contains a RNA binding motif. (GenBank Acc. No. CAA97170.1).

[0485] YHL035c: Is a known and non-essential protein (GenBank Acc. No.AAB65047.1). It is a member of the ATP-binding cassette superfamily.

[0486] YKL018w is also an essential protein containing a WD40 domainwhich is a typical protein binding domain. (GenBank Acc. No. CAA81853.1)

[0487] YLR221c: Is a protein of unknown function (GenBank Acc.No.AAB67410.1)

[0488] YML030w: Is a protein of unknown function (GenBank Acc. No.CAA86625.1)

[0489] YOR179c shows significant sequence similarity to Ysh1 (GenBankAcc. No. CAA99388.1)

[0490] Two further proteins for which binary interactions with membersof the polyadenylation complex as known so far have been shown beforehave also been purified with the complex:

[0491] YKL059c: is the product of an essential gene and is a zincbinding protein containing a C2HC Zinc finger. The presence of thisdomain predicts a RNA binding function of YKL059c. We believe thecorresponding gene product is identical to Pfs1, a protein which hasbeen mentioned in several publications, but which has never beenannotated in the databases (for review see Keller, W. andMinvielle-Sebastia (1997). Curr Opin Cell Biol 11: 352-357). (GenBankAcc. No. CAA81896.1)

[0492] Tif4632: Is a known and non-essential protein (GenBank Acc. No.CAA96751.1) which has been shown to have an RNA-binding/translationfactor activity and is involved in protein synthesis.

[0493] Below is a description of the experimental steps and protocols asused herein:

[0494] The initial round of purification of the complex was carried outusing Pta-1 as a bait as described below:

[0495] Construction of a Yeast Strain Expressing Tap-Tagged Pta1

[0496] The construction of these strains is illustrated both in FIG. 2and table 5.

[0497] Purification of Proteins Associated with PTA1

[0498] The TAP-technology, which is more fully described in WO/0009716and in Rigaut, G. et. al. (1999), Nature Biotechnology. Vol. 17 (10):1030-1032 respectively was used for protein complex purification. ThePta1 protein was C-terminally tagged with a TAP-tag which consists ofcalmodulin-binding peptide (CBP), a cleavage site for TEV proteasefollowed by two IgG-binding units of protein A (Rigaut, G. et. al.(1999), Nature Biotechnology. Vol. 17 (10): 1030-1032). Pta1 is anessential protein which has been reported to be a component of PFI.Pta1-TAP was used as a bait to identify associated partners from celllysates using the two-step TAP purification procedure. Proteins wereseparated by 1D gel electrophoresis and visualized by staining withCoomassie. More than a total of 20 bands could be detected on the gel(see FIG. 3). The identity of the proteins was determined by massspectrometry. 13 of these are known components of the pre-mRNAprocessing machinery: Cft1, Cft2, Ysh, Pta1, Rna14, Pab1, Pcf11, Pap1,Clp1, Pfs2, Fip1, Rna15 and Yth1. It is to be noted that such acomprehensive number has never before been purified together in form ofa complex. The remaining seven proteins have not previously been foundassociated with Pta1: Ref2, YK059c, YGR156w, YKL018w, Glc7, Ssu72 andYOR179c.

[0499] Validation of Interactions Found with Pta1

[0500] A reciprocal experiment to the one described above was performed.For this purpose a subset of the interactors found in the abovedescribed Pta purification (both known and novel interactors) werechosen as a bait for a further round of purification (the baits usedherein are listed in the first column of Table 1). In the case of someproteins the C-terminally tagged versions could not be recovered. Thelikely reason for this is that the addition of the TAP tag at theC-terminus interferes with the function of these proteins. An importantfact is that almost all of the known components involved in 3′-endformation and five of the seven novel proteins identified herein areessential for cell viability. The protein pattern obtained in some ofthose experiments is shown in FIG. 4. The construction of the strainswas carried out as described for the strain expressing the TAP-taggedPta-1.

[0501] Sequence Analysis of Members of the Complex

[0502] The process of mRNA processing is highly conserved in eukaryots.Accordingly, for a number of the yeast proteins human orthologues couldbe found (see Table 2). This illustrates that many of the functionsfound in the yeast complex can be transferred to humans. Also theenzymatic activity of this complex has long been known, theenzymatically active member could not yet be unraveled. Using extensivesequence similarity searches it could be shown that Ysh1 is homologousto a class of bacterial beta-lactamases. The active center of thisprotein family contains 2 zinc ions which are bound by histidines. Asthese residues are conserved in Ysh1 and it was shown that enzymaticactivity of the yeast complex is zinc dependent predicted that Ysh1 isresponsible for the catalytic activity of the complex. Two otherproteins found in the complex, Cft2 and YOR179c, are homologous to theYsh1 N- and C-terminus, respectively. Though Cft2 is homologous to theenzymatic region of Ysh1 it misses the zinc binding histidinesindicating that it lacks enzymatic activity. Thus, Cft 2 and YOR179ccould compete with Ysh1 for the same binding slot of the complex,suggesting a novel type of regulation of polyadenylation. A similar wayof regulation might be used in the case of Pfs2 and YKL018w, which bothconsist of multiple WD40 domains.

[0503] Prediction of Mammalian Proteins

[0504] To allow the transfer of function information from yeast to humanproteins, we did not only use an identity cutoff, but also the‘orthology’ concept. Orthology defines genes which arose via aspeciation event, in contrast to genes which arose via gene duplication.Orthologue genes are supposed to perform the same function in differentorganisms, therefore more detailed function information can betransferred. The algorithm for the detection of orthologous gene pairsfrom yeast and human uses the whole genome of these organisms. First,pairwise best hits were retrieved, using a full Smith-Waterman alignmentof predicted proteins. To further improve reli-ability, these pairs wereclustered with pairwise best hits involving Drosophila melanogaster andCaenorhabditis elegans proteins. See “Initial sequencing and analysis ofthe human genome”, Nature 2001 Feb 15; 409(6822):860-921 for a detaileddescription of the analysis.

[0505] Bioinformatic Analysis of the Complex:

[0506] Functional domains of all members of the complex were analyzedusing SMART (SMART: a web-based tool for the study of genetically mobiledomains. Nucleic Acids Res 2000 Jan 1; 28(1):231-4) and Pfam (Pfam:protein families database, Nucleic Acids Res 2000 Jan 1; 28(1):263-6).

[0507] Comparison of the Yeast and Mammalian Cleavage/PolyadenylationMachinery

[0508] The sequence of many of the polypeptides involved in 3′-endformation are conserved form yeast to mammals, although the sequenceelements on the substrate pre-mRNA differ (see FIG. 1).

[0509] The detailed experimental protocols for the example stated hereinare given below:

Protocols

[0510] Isolation of Protein Complexes:

[0511] a) Isolation FO Complexes from Yeast:

[0512] Yeast Strain Construction:

[0513] Yeast strains expressing TAP-tagged ORFs were constructed in asemi-automated way essentially according to Rigaut et. al. (Rigaut, G.et. al. Nat Biotechnol 17, 1030-2 (1999)) and Puig et al. (Puig, O. etal. Methotds 24, 218-19. (2001)) (See also FIG. 2 and Table 5)

[0514] TAP-purification using the Pta-1-tagged Strains:

[0515] Pta1-tagged strain was cultured in 4 l of YPD medium to an OD600of 2.

[0516] After harvesting, the cell pellet was frozen in liquid nitrogenand stored at −80° C. All further manipulations were done at 4° C.except where noted. For preparation of protein lysates the cells wereresuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.15%NP-40, 1.5 mM MgCl2, 0.5 mM DTT, protease inhibitors) and subjected tomechanical disruption with glass beads. Lysates were clarified by twosuccessive centrifugation steps at 20.000×g for 10 min and 100.000×g for1 hour. After addition of glycerol to 5% final concentration the lysateswere frozen in liquid nitrogen and stored at −80° C.

[0517] For the first purification step 500 μl of rabbit IgG-Agarose(50:50 slurry, Sigma A2909) pre-equilibrated in lysis buffer were addedto the lysate and the sample was rotated for 2 hours. The unboundfraction was discarded and the beads with the bound material weretransferred to a 0.8 ml column (MoBiTec M1002, 90 μm filter). The beadswere washed with 10 ml of lysis buffer followed by 5 ml of TEV cleavagebuffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% NP-40, 0.5 mM EDTA, 1mM DTT).

[0518] 150 μl of TEV cleavage buffer and 4 μl of TEV protease were addedto the column and the sample was incubated on a shaker at 16° C. for 2hours. The eluate was recovered by pressing with a syringe.

[0519] 150 μl of Calmodulin dilution buffer (10 mM Tris-HCl pH 8.0, 100mM NaCl, 0.1% NP-40, 2 mM MgAc, 2 mM imidazole, 4 mM CaCl2, 1 mM DTT)was added to the previous eluate and this mixture was transferred to aMoBiTec column containing 300 μl (bead volume) of Calmodulin affinityresin (Stratagene #214303) which was prewashed in Calmodulin wash buffer(10 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% NP-40, 1 mM MgAc, 1 mMimidazole, 2 mM CaCl2, 1 mM DTT). The samples were rotated for 1 hour at4° C.

[0520] After washing of the beads with 10 ml of Calmodulin wash buffer,protein complexes were eluted with 600 μl of elution buffer (10 mMTris-HCl pH 8.0, 5 mM EDTA). The samples were concentrated insiliconised tubes in a speed vac to a final volume of 10-20 μl. Proteinswere detected by polyacrylamide gel electrophoresis followed by stainingwith colloidal Coomassie blue.

[0521] General TAP-Purification Protocol for Soluble Proteins:

[0522] TAP-Purification of Soluble Proteins:

[0523] The purification was done from 2 liters of yeast cells grown tolate log phase (OD₆₀₀ ˜3-4). Cells were harvested and the pellet wasfrozen in liquid nitrogen and stored at −80° C. All steps were done at4° C. For preparation of protein lysates the cells were resuspended inbuffer A (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.15% NP-40, 1.5 mM MgCl₂,0.5 mM DTT, protease inhibitors) and subjected to mechanical disruptionwith glass beads. Lysates were clarified by two successivecentrifugation steps at 20.000×g for 10 min and 100,000×g for 1 hour.After addition of glycerol to 5% final concentration the lysates werefrozen in liquid nitrogen and stored at −80° C.

[0524] For the first purification step 500 μl of rabbit IgG-Agarose(50:50 slurry, Sigma A2909) pre-equilibrated in buffer A were added tothe lysate and the sample was rotated for 1 hour. The unbound fractionwas discarded and the beads with the bound material were transferred toa 0.8 ml column (MoBiTec M1002, 90 μm filter). The beads were washedwith 10 ml of buffer A.

[0525] 150 μl of buffer A and 4 μl of TEV protease (1 mg/ml) were addedto the column and the sample was incubated on a shaker at 16° C. for 1hour. The eluate was recovered by pressing with a syringe.

[0526] 150 μl of buffer A containing 4 mM CaCl₂ was added to theprevious eluate and this mixture was transferred to a MoBiTec columncontaining 300 μl (bead volume) of Calmodulin affinity resin (Stratagene#214303) which was prewashed in buffer A containing 2 mM CaCl₂. Thesamples were rotated for 1 hour at 4° C.

[0527] After washing of the beads with 5 ml of buffer A containing 2 mMCaCl₂, protein complexes were eluted with 600 μl of elution buffer (10mM Tris-HCl pH 8.0, 5 mM EGTA). The samples were concentrated insiliconized tubes in a speed vac. Proteins were detected bypolyacrylamide gel electrophoresis followed by staining with colloidalCoomassie blue.

[0528] TAP-Purification of Membrane Proteins:

[0529] The purification was done from 2 liters of yeast cells grown tolate log phase (OD₆₀₀ ˜3-4). Cells were harvested and the pellet wasfrozen in liquid nitrogen and stored at −80° C. All steps were done at4° C. For the purification of TAP-tagged membrane proteins cells werelysed in buffer containing 50 mM Hepes/KOH pH 7.5, 150 mM KCl, 0.25%NP40, 2 mM MgCl₂, 2 mM EDTA, 0.5 mM DTT and protease inhibitors. Theextracts were spun at 20,000×g for 10 min and the resulting supernatantwas adjusted to 1.5% NP-40 and 5% glycerol. Samples were incubated for30 min with end-over-end shaking and then centrifuged at 180,000×g for30 min. The resulting supernatant was immediately used forTAP-purification.

[0530] For the first purification step 500 μl of rabbit IgG-Agarose(50:50 slurry, Sigma A2909) pre-equilibrated in buffer B (50 mM Tris-HClpH 7.5, 100 mM NaCl, 0.5% NP-40, 1.5 mM MgCl₂, 0.5 mM DTT, proteaseinhibitors) was added to the lysate and the sample was rotated for 1hour. The unbound fraction was discarded and the beads with the boundmaterial were transferred to a 0.8 ml column (MoBiTec M1002, 90 μmfilter). The beads were washed with 10 ml of buffer B.

[0531] 150 μl of buffer B and 8 μl of TEV protease (1 mg/ml) were addedto the column and the sample was incubated on a shaker at 16° C. for 1hour. The eluate was recovered by pressing with a syringe.

[0532] 150 μl of buffer B containing 4 mM CaCl₂ was added to theprevious eluate and this mixture was transferred to a MoBiTec columncontaining 300 μl (bead volume) of Calmodulin affinity resin (Stratagene#214303) which was prewashed in buffer B containing 2 mM CaCl₂. Thesamples were rotated for 1 hour at 4° C. After washing of the beads with5 ml of buffer B containing 2 mM CaCl₂, protein complexes were elutedwith 600 μl of elution buffer (10 mM Tris-HCl pH 8.0, 5 mM EGTA). Thesamples were concentrated in siliconized tubes in a speed vac. Proteinswere detected by polyacrylamide gel electrophoresis followed by stainingwith colloidal Coomassie blue.

[0533] b) Isolation of Complexes from Mammalian Cells

[0534] Cells:

[0535] Retroviral transduction vectors containing the TAP-cassette weregenerated by directional cloning of PCR-amplified ORFs into a modifiedversion of a MmoLV-based vector via the Gateway site-specificrecombination sstem (Life Technologies). Virus stocks were generated ina HEK293 gag-pol packaging cell line by pseudotyping with VSV-G. Cellswere infected and complexe were purified after cell expansion andcultivation of 3-5 using a modified TAP-protocoll

[0536] Standard Lysis Protocol:

[0537] The medium was removed from the culture dish and the cells werescraped directly from the plate with help of a rubber policeman. Thecells were collected on ice washed 3 times with PBS and resuspended inlysis buffer (50 mM Tris, pH: 7.5; 5% glycerol; 0,2% IGEPAL; 1.5 mMMgCl2; 1 mM DTT; 100 mM NaCl; 50 mM NaF; 1 mM Na3VO4+proteaseinhibitors). The cells were lysed for 30 min on ice, spun for 10 min. at20,000 g and re-spun for 1 h at 100,000 g. The supernatant wasrecovered, rapidly frozen in liquid nitrogen and stored at −80° C. Forpre-clearing the thawed lysate was incubated with 500 μl sepharose CL-4Bbeads (Amersham Pharmacia) for 1 h shaking and finally processedaccording the TAP protocol.

[0538] Nuclear Lysis Protocol:

[0539] The medium was removed from the culture dish and the cells werescraped directly from the plate with help of a rubber policeman. Thecells were collected on ice washed 3 times with PBS and resuspended inbuffer A (10 mM Tris-Cl, pH 7.5; 1, 5 mM MgCl2; 10 mM KCl; 1 mM DTT, 50mM NaF; 1 mM Na3VO4). To isolate the nuclei the lysate was dounced witha tight fitted pestle in a dounce homogenizer for 15 strokes. The nucleiwere harvested by centrifugation (10 min. at 2000 g and 20 min. at16,000 g) and lysed in buffer B (50 mM Tris-Cl, pH: 7.5; 1.5 mM MgCl;20% glycerol; 420 mM NaCl; 1 mM DTT; 50 mM NaF; 1 mM Na3VO4) for 30 min.on ice with frequent shaking. The protein lysate was cleared bycentrifugation (30 min at 100,000 g) and 1:4 diluted with buffer C (50mM Tris-Cl, pH: 7.5; 1 mM DTT; 0.26% NP40; 1.5 mM MgCl; 50 mM NaF; 1 mMNa3VO4). After 30 min incubation on ice the lysate was re-spun for 30min at 100,000 g, quickly frozen in liquid nitrogen and stored at −80°C. For pre-clearing the thawed lysate was incubated with 500 μlsepharose CL-4B beads (Amersham Pharmacia) for 1 h shaking and finallyprocessed according the TAP protocol.

[0540] Mass Spectrometric Analysis

[0541] Protein Digestion Prior to Mass Spectrometric Analysis:

[0542] Gel-separated proteins were reduced, alkylated and digested ingel essentially following the procedure described by Shevchenko et al.(Shevchenko, A., Wilm, M., Vorm, O., Mann, M. Anal Chem 1996, 68,850-858). Briefly, gel-separated proteins were excised from the gelusing a clean scalpel, reduced using 10 mM DTT (in 5 mM ammoniumbicarbonate, 54° C., 45 min) and subsequently alkylated with 55 mMiodoacetamid (in 5 mM ammonium bicarbonate) at room temperature in thedark (30 min). Reduced and alkylated proteins were digested in gel withporcine trypsin (Promega) at a protease concentration of 12.5 ng/μl in 5mM ammonium bicarbonate. Digestion was allowed to proceed for 4 hours at37° C. and the reaction was subsequently stopped using 2 μl 25% TFA.

[0543] Desalting and Concentration of Peptides Produced By In-GelDigestion of Gel-Separated Proteins:

[0544] Peptides were desalted and concentrated using a prefabricateduZipTip (Millipore) reversed phase column. Peptides were eluted directlyonto stainless steel MS sample holders using 2 μl eluent (70%acetonitrile in 5% TFA containing 2 mg/ml alpha-Cyano-4-hydroxy-cinnamicacid and two standard peptides for internal calibration of massspectra).

[0545] Mass Spectrometric Data Acquisition:

[0546] Matrix-assisted laser desorption/ionisation (MALDI)time-of-flight (TOF) mass spectra were acquired in delayed extractionmode on a Voyager DE-STR PRO MALDI mass spectrometer (AppliedBiosystems) equipped with a 337 nm nitrogen laser. 500 laser shots wereaveraged in order to produce final spectra. Spectra were automaticallyinternally calibrated using two standard peptides. The monoisotopicmasses for all peptide ion signals detected in the acquired spectra weredetermined and used for database searching.

[0547] Protein Sequence Database Searching Using Peptide MassFingerprinting (PMF) Data:

[0548] The list of monoisotopic peptide masses obtained from the MALDImass spectrum was used to query a fasta formatted protein sequencedatabase that contained all protein sequences from S. cerevesia.Proteins were identified by peptide mass fingerprinting (Mann, M.,Højrup, P., Roep-storff, P. Biol Mass Spectrom 1993, 22, 338-345;Pappin, D., Højrup, P, Bleasby, AJ Curr. Biol. 1993, 3, 327-33; Henzel,W. J., Billeci, T. M., Stults, J. T., Wong, S. C., Grimley, C.,Watanabe, C. Proc Natl Acad Sci U S A 1993, 90, 5011-5015; Yates, J. R.,Speicher, S., Griffin, P. R., Hunkapiller, T. Anal Biochem 1993, 214,397-408; James, P., Quadroni, M., Carafoli, E., Gonnet, G. BiochemBiophys Res Commun 1993, 195, 58-64) using the software tool Profound(Proteometrics). In PMF, a protein is identified by correlating themeasured peptide masses with theoretical digests of all proteins presentin the database. Search criteria included: tryptic protein cleavage,monoisotopic masses, 30 ppm mass accuracy. No restrictions on proteinsize or isoelectric point were imposed.

[0549] Bioinformatics

[0550] Functional and localization information about yeast proteins wasretrieved from the Yeast Protein Database (YPD (Constanzo, M. C. et al.,2001, Nucl. Acid Res, 29: 75-9; Hodges, P. E. et al., 1999, Nucl. AcidsRes 27: 69-73)) released in August 2001. In order to get a more conciseclassification for localization and function, YPD classes were merged.Protein domain analysis was performed using SMART (Schultz, J., Copley,R. R., Doerks, T., Ponting, C. P. & Bork, P. Schultz, J., Copley, R. R.,Doerks, T., Ponting, C. P. & Bork, P. SMART, Nucleic Acids Res 28,231-4. (2000)). PsiBlast (Altschul, S. F. et al. Gapped BLAST andPSI-BLAST: Nucleic Acids Res 25, 3389-402. (1997)) was used for homologyanalysis. All additional analysis software has been developed in house,using Perl and Python.

Assays for Assaying the Activities of the Complexes Presented in theInvention

[0551] An exemplary RNA binding assay can be carried out by contacting acomplex having RNA binding activity with a radioactive [32P] end-labeledRNA substrate, e.g. a poly (A) RNA, under appropriate conditions anddetecting bound protein. The detection of bound protein can be carriedout, e.g., by filtrating the solution through a nitrocellulose filterand determining the radioactivity bound to the filter. This assay isbased on the retention of nucleic acid-protein complexes onNitrocellulose whereas free nucleic acid can pass through the filter

[0552] (see e.g. Wahle, E., 1991, Methods 66: 759-68)

[0553] An exemplary RNA exonuclease assay can be carried out bycontacting a complex having RNA exonuclease activity with aradioactivity [32 phosphate] end-labeled RNA substrate under appropriateconditions and detecting the release of free radioactive nucleotides.The detection of free radioactive nucleotides can be carried out, e.g.,by adding 20% trichloroacetic acid, filtrating the solution through afilter and measuring the amount of acid-soluble radioactivity

[0554] (see e.g. Ross, J., 1999, Methods 17: 52-9)

[0555] An exemplary mRNA splicing assay can be carried out by contactinga complex having mRNA splicing activity with a radioactively labeled RNAsubstrate under appropriate conditions and detecting the release ofspliced RNA species. The detection of spliced RNA species can be carriedout, e.g., by fractionation of processed RNAs in a glycerol gradient andsubsequent analysis by denaturing polyacrylamide gel elecrophoresis andvisualization by autoradiography.

[0556] (see e.g. Schwer, B. and Gross, C H., 1998, Methods17: 2086-94)

[0557] An exemplary rRNA processing assay can be carried out bycontacting a complex having rRNA processing activity with an pre-rRNAsubstrate under appropriate conditions and detecting the release of freeprocessed rRNA species. The detection of processed rRNA species can becarried out, e.g., using a primer extension or northern blotting assayby measuring the size of the rRNA species.

[0558] (see e.g. Kressler, D. et al, 1997, Methods 17: 7283-94) ColumnCOLUMN A: COLUMN C D: known Cleavage/ Proteins components COLUMN B:poly- of Entry Proteins of the novel adenylation Activity of unknownpoint Interactions found complex proteins machinery the complex functionlocal. Cft1 Cft1, Cft2, Pta1, Act1 Cft1 Cft1 Cft2 Clp1 Act1 Cka1 Act1Cft1 Cft2 3′ end Ycl046w c n u e Cft2 Ysh1 coippt; Cft2 Cka1 Fip1 Pab1Eft2 Eno2 Cka1 Clp1 processing Ygr156w Clp1 Cft1, Cft2, Fip1, Clp1 Eft2Pap1 Pcf11 Glc7 Gpm1 Eft2 Eno2 activity for Yhl035c Fip1 Pap1, Pta1,Eno2 Fip1 Pfs2 Pta1 Hhf2 Hta1 Fip1 Glc7 mRNA Ykl018w Pap1 Ykl059c, Ysh1,Glc7 Rna14 Rna15 Hsc82 Imd2 Gpm1 Hhf2 Ylr221c Pcf1 Yth1 coippt; Gpm1Tif4632 Imd4 Met6 Hsc82 Hta1 Yml030w 1 Fip1, Yth1 2- Hhf2 Ykl059c Ysh1Pdc1 Pfk1 Imd2 Imd4 Yor179c Pfs2 hybrid, coippt, Hsc82 Yth1 Ref2 Sec13Met6 Pab1 Pta1 high-throughput Hta1 Imd2 Sec31 Ssa3 Pap1 Pcf11 Ref22-hybrid, in vitro Imd4 Met6 Ssu72 Taf60 Pdc1 Pfk1 Rna1 binding; Glc7,Pab1 Tkl1 Tsa1 Pfs2 Pta1 4 Ref2; Fip1, Pap1 Tye7 Vid24 Ref2 Rna14 Rna1Rna14 2-hybrid; Pcf11 Vps53 Rna15 Sec13 5 Rna14, Rna15 Pdc1 Pfk1 Ycl046wSec31 Ssa3 Ykl0 2-hybrid; Clp1, Pfs2 Pta1 Ygr156w Ssu72 Taf60 59c Pcf11coippt, Ref2 Yhl035c Tif4632 Tkl1 Yor1 high-throughput Rna14 Ykl018wTsa1 TYe7 79c 2-hybrid; Fip1, Rna15 Ylr221c Vid24 Vps53 Ysh1 Pap12-hybrid, Sec13 Yml030w Ycl046w Yth1 coippt; Fip1, Sec31 Yor179c Ygr156wYsh1 coippt, Ssa3 Yhl035c copurification; Ssu72 Ykl018w Hhf2, Hta1 Taf60Ykl059c affinity column, Tif4632 Ylr221c coippt; Sec13, Tkl1 Tsa1Yml030w Sec31 2-hybrid, TYe7 Yor179c Ysh1 affinity column, Vid24 Yth1copurification, Vps53 high-throughput Ycl046w 2-hybrid; Pab1, Ygr156wPcf11p, Yhl035c Rna14p, Ykl018w Rna15p Ykl059c copurification; Ylr221cPab1, Tif4632 Yml030w copurification, in Yor179c vitro binding; Ysh1Yth1 Pcf11, Rna14, Rna15 copurification; Pcf11, Rna14 2- hybrid, coippt,copurification, high-throughput 2-hybrid; Pcf11, Rna15 2-hybrid,copurification, high-throughput 2-hybrid; Pfs2, Rna14 2-hybrid, coippt;Pfs2, Ysh1 coippt; Fip1, Pfs2 coippt; Pap1, Yth1 coippt; Clp1, Rna14coippt; Clp1, Rna15; Ysh1, Yth1 in vitro binding; Sec13, Sec31 CoatomerCOPII complex; Cft1, Cft2, Pta1, Ysh1 Pre- mRNA cleavage factor II;Hhf2, Hta1 Histone octamer; Cft1, Cft2, Fip1, Pap1, Pta1, Ykl059w, Ysh1,Yth1 Polyadenylation Factor I (PFI); Cft1, Cft2, Fip1, Pap1, Pfs2, Pta1,Ykl059w, Ysh1, Yth1 polyadenylation factor I (PF I); Clp1, Pab1, Pcf11,Rna14, Rna15 Pre- mRNA cleavage and polyadenylation factor IA; Fip1,Pap1, Ysh1 Pre-mRNA polyadenylation factor I; Sec13, Sec31 Sec13p-Sec31p

[0559] TABLE 2 INDIVIDUAL YEAST PROTEINS OF THE COMPLEXES A) Yeastproteins Human C. elegans Drosophila listed in SEQ SWISS- orthologueorthologue orthologue table 2 ID MIPS PROT SGD Genbank in GenBank inGenBank in GenBank ACT1 1 ATBY P02579 S0001855 D50617 gi4501885ACT4_CAEE Q9W460 L CFT1 3 S61187 S0002709 U28374 CFT2 5 S64952 S0004105Z73287 CKA1 7 A31564 P15790 S0001297 Z46861 CLP1 9 S67147 S0005776Z75158 gi5803029 YMI4_CAEE Q9V6Q1 L EFT2 11 P32324 S0002793 AAB64827.1gi: 4503483 EF2_(—) CAEEL Q9V9RO ENO2 13 NOBY2 P00925 S0001217 U00027gi: 4503571 ENO_CAEE Q9VQ38 L GLC7 15 S32595 P32598 S0000935 U18916gi4506003 YME1_CAE Q9VC69 EL GPM1 17 PMBYY P00950 S0001635 Z28152 Fip119 A56545 P45976 S0003853 Z49593 HHF2 21 P02309 S0004975 Z71306 HTA1 23HSBYA1 P04911 S0002633 Z48612 HSC82 25 S55133 P15108 S0004798 CAA89919.1gi6680307 gi3875041 gi7292327 IMD2 27 S48997 P38697 S0001259 AAB69728.1gi4504689 gi18030187 gi7291188 IMD4 29 S50890 P50094 S0004520 CAA86719.1gi4504688 gi18030187 gi7291188 MET6 31 S50594 P05694 S0000893 AAB64646.1PAB1 33 DNBYPA P04147 S0000967 U18922 F15P000000 Q9U302 Q9V8C3 84557PAP1 35 S19031 P29468 S0001710 Z28227 P51003 Q20370 Q9V8X7 PCF11 37S59435 P39081 S0002636 Z48612 gi7706224 YRR2_CAEE Q9V768 L PDC1 39 DCBYPP06169 S0004034 Z73216 PFK1 41 JQ0016 P16861 S0003472 Z73025 PFS2 43S51295 P42841 S0005261 Z71593 F15P000000 CAB76722 Q9VNG2 84546 PTA1 45S31299 Q01329 S0000041 U12980 REF2 47 S52702 P42073 S0002603 Z48784RNA14 49 S54561 P25298 S0004665 Z49703 RNA15 51 B40257 P25299 S0003012Z72566 F15P000000 O45577 Q9VE52 61276 SEC13 53 A45442 Q04491 S0004198U14913 P55735 AAF36082 Q9V3J4 SEC31 55 S58782 P38968 S0002354 Z74243gi7662370 O45604 Q9V4Z0 SSA3 57 S36753 P09435 S0000171 Z35836 gi5729877Q93601 Q9VFB0 SSU72 59 S63180 P53538 S0005166 Z71498 TAF60 61 S64120P53040 S0003080 Z72634 gi5032147 O17279 Q9VW16 TIF4632 63 B48086 P39936S0003017 Z72571 TKL1 65 XJBYTK P23254 S0006278 Z49219 gi4507521 O17759Q9VHN7 TSA1 67 A47362 P34760 S0004490 Z46659 TYE7 69 S48252 P33122S0005871 Z75252 VID24 71 S48270 P38263 S0000309 Z35974 VPS53 73 S56801P47061 S0003566 Z49304 F15000000 YNP8_CAEE Q9VQY8 87184 L YSH1 75 S51413S0004267 U17245 YTH1 77 S59772 S0006311 U32445 YCL046W 79 S19375 P25575S0000551 X59720 YGR156W 81 S60446 P39927 S0003388 YHL035C 83 S48933P38735 S0001027 U11583 YKL018W 85 S37831 P36104 S0001501 Z28018F15P000000 Q18403 Q9VLN1 64108 YLR221C 87 S51444 S0004211 U19027 YKL059C89 S37881 P35728 S0001542 Z28059 YML030W 91 S49749 Q03713 S0004492Z46659 YOR179C 93 S67071 S0005705 Z75087

[0560] B) Yeast proteins Biochemical listed in function Cellularfunction table 2 YPD description from YPD from YPD ACT1 Actin, involvedin cell Structural protein Cell polarity; Cell polarization,endocytosis, structure; and other cytoskeletal Chromatin/chromosomefunctions structure; Mating response; Pol II transcription; Vesiculartransport CFT1 Component of pre-mRNA Hydrolase; Nuclease RNA cleavagefactor II [endo, exo, ribo, processing/modification deoxyribo] CFT2Component of pre-mRNA RNA-binding protein RNA cleavage factor IIprocessing/modification CKA1 Casein kinase II (protein Protein kinase;Pol III transcription kinase CK2), catalytic Transferase (alpha) subunitCLP1 Subunit of cleavage and Nuclease [endo, RNA polyadenylation factorIA, exo, ribo, deoxyribo] processing/modification required for 3′-endprocessing of pre-mRNA EFT2 Translation elongation Translation factorProtein synthesis factor EF-2, identical to Eft1p, contains diphthamidewhich is not essential for its activity ENO2 Enolase 2(2-phosphoglycerate Lyase Carbohydrate metabolism dehydratase); converts2-phospho-D- glycerate to phosphoenolpyruvate in glycolysis FIP1Component of polyadenylation RNA polymerase RNA factor that interactswith poly(A) subunit; RNA- processing/modification polymerase bindingprotein; Regulatory subunit GLC7 Protein serine/threonine phosphataseHydrolase; Protein Carbohydrate PP1 required for glucose phosphatasemetabolism; Cell polarity; repression, membrane bilayer mixing, Cellstress; Meiosis; and ER-to-Golgi and endocytic Mitosis vesiculartrafficking GPM1 Phosphoglycerate mutase that Isomerase Carbohydrateconverts 2-phosphoglycerate to 3- metabolism; Energy phosphoglycerate inglycolysis generation HHF2 Histone H4, identical to Hhf1p DMA-bindingprotein Chromatin/chromosome structure; Pol I transcription HTA1 HistoneH2A, nearly identical DMA-binding protein Cell stress; to Hta2pChromatin/chromosome structure; Pol II transcription HSC82 Chaperoninhomologous to E. coli Heat shock protein; Protein folding; Cell stressHtpG and mammalian HSP90 Hydrolase; ATPase; Chaperones IMD2Inosine-5′-monophosphate Oxidoreductase Nucleotide metabolismdehydrogenase, catalyzes the conversion of inosine 5′-phosphate andNAD(+) to xanthosine 5′-phosphate and NADH, the first reaction unique toGMP biosynthesis IMD4 Protein with similarity to OxidoreductaseNucleotide metabolism inosine-5′-monophosphate dehydrogenase MET6Homocysteine methyltransferase Transferase Amino-acid metabolism(5-methyltetrahydropteroyl triglutamate-homocysteine methyltransferase),methionine synthase, cobalamin- independent PAB1 Poly(A)-binding proteinof RNA-binding protein; Protein synthesis; RNA cytoplasm and nucleus,part Translation factor processing/modification; of the 3′-end RNA- RNAturnover processing complex (cleavage factor I), has 4 RNA recognition(RRM) domains PAP1 Poly(A) polymerase, required RNA polymerase RNA formRNA 3′ end formation, subunit; RNA- processing/modification has apoorly conserved RNA binding protein; recognition (RRM) domainTransferase PCF11 Component of pre-mRNA cleavage Nuclease [endo, RNA andpolyadenylation factor I, exo, ribo, deoxyribo]; processing/modificationinteracts with Rna14p and Rna15p RNA-binding protein PDC1 Pyruvatedecarboxylase isozyme 1 Lyase Carbohydrate metabolism PFK1Phosphofructokinase alpha subunit, Other kinase; Carbohydrate metabolismpart of a complex with Pfk2p which Transferase catalyze ATP-dependentconversion of fructose-6-phosphate to fructose- 1,6-bisphosphate, a keyregulatory step in glycolysis PFS2 Polyadenylation factor I UnknownProtein complex subunit 2 required for mRNA assembly; RNA 3′-endprocessing, processing/modification bridges two mRNA 3′-end processingfactors, has WD (WD-40) repeats PTA1 Component of pre-mRNA cleavageHydrolase; Nuclease RNA factor II (CFII), required for [endo, exo, ribo,processing/modification both cleavage and polyadenylation deoxyribo] ofmRNA precursor REF2 Protein involved in mRNA RNA-binding protein RNA3′-end formation before processing/modification polyadenylation, mutantdisplays significantly lower usage of weak poly(A) sites RNA14 Componentof pre-mRNA cleavage Hydrolase; Nuclease RNA and polyadenylation factorI [endo, exo, ribo, processing/modification (CFI) involved in poly(A)site deoxyribo] choice, interacts with Rna15p, Fip1p, Pap1p, and Pcf11pRNA15 Component of pre-mRNA cleavage Nuclease [endo, RNA andpolyadenylation factor I exo, ribo, deoxyribo]; processing/modification(CFI), involved in poly(A) site RNA-binding protein choice, interactswith Rna14p, Pap1p, and Pcf11p, contains one RNA recognition (RRM)domain SEC13 Component of the COPII coat of Unknown Small moleculetransport; vesicles involved in endoplasmic Vesicular transportreticulum to Golgi transport, contains six WD (WD-40) repeats SEC31Component (p150) of the COPII Vesicle coat protein Vesicular transportcoat of secretory pathway vesicles involved in endoplasmic reticulum toGolgi transport, associated with Sec13p, member of WD (WD-40) repeatfamily SSA3 Chaperone of the HSP70 family, Chaperones; Heat Cell stress;Protein heat-induced cytoplasmic form not shock protein folding; Proteinexpressed under optimal conditions translocation SSU72 Protein thatinteracts with Complex assembly Pol II transcription TFIIB (Sua7p) andinfluences protein RNA polymerase II start-site selection in sua7mutants TAF60 Component of TAF(II) complex Transcription factor Pol IItranscription (TBP-associated protein complex) and SAGA complex(Spt-Ada-Gcn5-acetyltransferase), required for activated transcriptionby RNA polymerase II TIF4632 mRNA cap-binding protein RNA-bindingprotein; Protein synthesis (elF4F) 130K subunit Translation factor TKL1Transketolase 1 Transferase Amino-acid metabolism; Carbohydratemetabolism TSA1 Thioredoxin peroxidase, abundant Oxidoreductase Cellstress thiol-specific antioxidant protein that prevents formation ofsulfur-containing radicals TYE7 Basic helix-loop-helix transcriptionTranscription factor Pol II transcription factor that can suppress theGcr1p requirement for glycolytic gene expression VID24 Protein requiredfor vacuolar import Unknown Protein degradation; and degradation ofFbp1p (fructose- Vesicular transport 1,6-bisphosphatase) VPS53 Subunitof the Vps52p-Vps53p-Vps54p Docking protein Vesicular transport complex,involved in protein sorting in the late Golgi YCL046W Protein of unknownfunction Unknown Unknown YGR156W Unknown Unknown YHL035C Member of theATP-binding ATP-binding Small molecule transport cassette (ABC)superfamily cassette; ATPase; Active transporter, primary; Hydrolase;Transporter YKL018W Protein of unknown function Unknown Unknown YLR221CProtein of unknown function Unknown Unknown YKL059C Protein withsimilarity to Unknown Unknown members of the chaperonin- containingT-complex YML030W Protein of unknown function, Unknown Energy generationmay be involved in mitochondrial translation YOR179C Protein withsimilarity to Ysh1p Unknown Unknown YSH1 Component of pre-mRNA cleavageRNA-binding protein RNA factor II (CFII), required forprocessing/modification processing of mRNA 3′ end YTH1 Component ofpolyadenylation Unknown RNA factor, required for bothprocessing/modification cleavage and polyadenylation of pre-mRNA

[0561] TABLE 3 MEDICAL APPLICATION OF THE COMPLEX Name of Cellularcomplex role Medical applications Poly- RNA infectious diseases; viraladenylation- processing/ infections such as herpes complex modifi-simplex infections, Epstein- cation Barr-infections, influenza;metabolic disease such as metachromatic leukodystrophy;neurodegenerative disorders such as amyotrophic lateral sclerosis;cancer

[0562] TABLE 4 CHARACTERIZATION OF PREVIOUSLY UNDESCRIBED PROTEINS Listof proteins of Motifs found predicted unknown by sequence knownfunctions Putative function analysis orthologues YCL046W — YGR156W RRMYHL035C multispecific organic 3x TM, no ortholog, anion transporter 1,2x PFAM: but high multidrug resistance- ABC-membrane identity toassociated protein 2) domain, MRP1-HUMAN 2x AAA (P33527), MRP3-HUMAN(O15438), MRP2-HUMAN (Q92887) YKL018W guanine nucleotide 4x WD40 bindingprotein YLR221C — YML030W 2x trans- Q9P297 (CLST membrane 11240 protein)YOR179C polyadenylation — Legend AAA ATPases associated with a varietyof cellular activities ABC-membrane unit of six transmembrane helices;many members of ABC domain transporter family have two such regions RRMRNA recognition motif WD40 40 residues repeat first identified in betatransducin

Table 5 Overview on Experimental Steps High Troughput Production of TAPTagged Yeast ORFs

[0563] 1) Oligo Design

[0564] The oligo are semi-automatically designed in FileMaker Pro. The3′ end of the ORF (forward oligo, 51 nucleotides), the 3′ UTR (100nucleotides used to design the reverse oligo) and sequence 500nucleotides upstream of the stop codon (40 nucleotides, check oligos)are directly extracted from database (SDG). For the reverse oligo, theprogram automatically gives the antiparallel sequence. The 17 constantnucleotide sequences used to prime the PCR reaction are automaticallyadded at the 3′ end of the oligo

[0565] 2) Target PCR

[0566] Fully Automated (Tecan Robot)

[0567] Materials: target plasmid: pBS1539 (described in Rigaut G, etal., Nat Biotechnol. 1999 Oct;17(10):1030-2.)

[0568] oligos: purchased from MWG, forwards and reverse primers arepre-mixed to a final concentration of 10 micromolar and delivered in a96 well plate format tempera- step volume ture Device prepare master upto 4 C. cooling mix (AmpliTaq, 2.5 ml master platform 1 Perkin Elmer),mix containing the TAP cassette vector (1.5 ng/25 microliter reaction)Dispense the 23.5 4 C. cooling master mix in 96 microliters masterplatforms well plate = mix + 1 + 2 reaction plate reaction (96 wellplate) plate Add 1.0 microliters 1.0 4 C. cooling of oligo (finalmicroliters reaction platform 2 concentration: 0.4 plate microM) toreaction plate Cycle (30 cycles) MJ the PCR reaction thermocyclersPipette 2 microliters 2 rt Pharmacia from the reaction microliters readyplate to a 96 well gel to run gels (Pharmacia) run gel 5 minutesPharmacia, Ready-To-Run

[0569] 3) Yeast Transformation

[0570] General considerations: Procedure partially automated

[0571] Materials: the haploid yeast strain is MGD453-13D: MATa, ade2,arg4, leu2-3,112, trp1-289, ura3-52. “novelties” verus tempera- originalstep volume ture device protocole. The day before, 200 ml 30 C. shakinginoculate 200 ml incubator of YPD with ˜100 microliters of freshlygrowing yeast culture When the culture 2 ml rt uses of 24 reaches anOD600 well plates of 1.0, dispense (Qiagen) 2 ml of yeast culture in 24well plate = transformation plate (4) spin heraus rt centrifuge aspiratethe sup 2 ml rt ressuspend cells rt shaker no washing in the remainingsteps of of media the cells dispense 5 5 rt microliters microliters ofcarrier DNA, (Herring testes Carrier DNA; 10 mg/ml; Clontech) mix rtshaker transfer 20 rt no cleaning remaining microliters of the PCR ofPCR from reaction reaction plate to the trans- formation plate mix rtshaker add 500 500 rt microliters microliters PEG/LiAc (viscous) mix rtshaker add 55 50 rt shaker microliters microliters DMSO, mix incubate15′ rt rt incubate 15′ 42 C. heating at 42 C. platform with custom madePelletier elements transfer at 700 rt rt + 700 microliters microlitersTE spin rt aspirate sup rt add 800 800 rt microliters microliters YPDincubate 30′ 30 C. heating at 30 C + platform + shake shaker add 1 ml ofrt TE spin rt aspirate sup rt add 50 50 rt 8 tips microlitersmicroliters TE mix rt shaker plate + incubate for ˜3 days at 30 C. (10cm Petri dishes or 12-24 wellplates)

[0572] 4) Check PCR

[0573] general considerations: fully automated. According to results ofthe transformation 0 to 6 colonies are tested for homologousrecombination. These results are filed in an excell file directly linkedto the robot program.

[0574] Material: the forward oligos are specific for each ORF (for tesequence cf 1); purchased from MWG at 10 micromolar in 96 well plates.The reverse oligo is constant for all ORFs and annealed in the TAPsequense dispense 20 20 microliters rt microliters NaOH (20 mM) in 96well plates pickup colonies (0 to 6) in the 96 well plate containing theNaOH = DNA plate (96 well) Boil 2′ add 140 microliters of TE pH 7.5 (8xdilution) spin at 2000 rpm Prepare master up to 9 ml microliters 4 C.cooling mix (PCR master, platform Roche) 1 dispense 14.5 14.5microliters 4 C. cooling microliters in platform reaction plate 2 (96well plate) add 2.5 2.5 microliters 4 C. cooling microliters reactionplatform of oligo (final plate 1 concentration 1 microM) to reactionplate transfer 8 8 microliters 4 C. cooling microliters reactionplatform DNA from DNA plate 1 plate to reaction plate Cycle (30 cycles)MJ the PCR reaction thermo- cyclers add 1 microliters 5 microliters rtof LB load 10 10 microliters Pharmacia microliters ready to on a 96 wellrun gels agarose gel run gel 5 Pharmacia, minutes Ready-To-Run

[0575] 5) Dot Blot Analysis the remaining of the check PCR 30 C.incubator positive colonies are restreacked on -ura plates. Plates areincubated at 30 C. the next day, the restreack is used to 30 C. shakerinoculate 2 ml of YPD in 24 well plates (Qiagen). Plates are incubatedover night at 30 C. Culture plates are spin remove the supernatant add100 microliters of waters mix shaker add 100 microliters of 0.2 M NaOHmix shaker incubate 3 minutes at room temperature spin remove thesupernatant add 50 microliters of 2× sample buffer boil 3 minutes spinload on a 96 dot blot apparatus Biodot, BioRad dot blot on anitrocellulose membranne Protran, Schleicher and Schuell detect TAPtagged protein by ECL using peroxidase anti-peroxidase complex

[0576] TABLE 6 KNOWN COMPONENTS OF THE YEAST mRNA 3′-END PROCESSINGMACHINERY Poly- peptide comp. Gene product/ Factor Function (kDa) (ORF)Sequence motifs CF IA Cleavage and 79.8 Rna14 8xHAT domainspolyadenylation 71.9 (YMR061w) 50.0 Pcf11 (YDR228c) 32.8 Clp1 (YOR250c)RRM Rna15 (YGL044c) Polyadenylation 64.2 4×RRM 1xPolyA Pab1 (YER165w) CFIB Cleavage site 73 Hrp1 (YOL123w) selection and polyadenylation CF II/Cleavage and 153.4 Cft1/Yhh1 (YDR301w) PF I polyadenylation 96.1Cft2/Ydh1 (YLR115w) Lactamase (= CPF) 87.6 Ysh1/Brr5 (YLR277c)NTP_transfer_2 88.3 Pta1 (YAL043c) 64.4 Pap1 (YKR002w) 7xWD40 Pfs15xZnF_C3H1 53.1 Pfs2 (YNL317w) 35.6 Fip1 (YJR093c) 24.4 Yth1 (YPR107c)

[0577] NOVEL COMPLEX MEMBERS Gene product Factor Function kDA (OKE) Seq.Motifs 59.7 Ref2 (YDR195w) — 59.5 YKL059c = Pfs1? ZnF_C2HC 46.8 YGR156wRRM 37.0 YKL018w 4xWD40 35.9 Glc7 (YER133w) PP2Ac 23.3 Ssu72 (YNL222w) —20.9 YOR179c similarity to Ysh1 Act1 Actin Cka1 Kinase Eft2 Eno2 EnolaseGpm1 Hhf2 Core Histone Hsc82 Hta1 Core Histone Imd2 Imd4 Met6 Pdc1 Pfk1Phosphofrukto- kinase Sec13 WD domain, G-beta repeat Sec31 WD domain,G-beta repeat Ssa3 Hsp70 protein domain Taf60 — Tif4632 Helix-loop-HelixDNA-bind. Tsa1 Tye7 — Vid24 — Vps53 — YCL046w ABC transporter transmembreg YHL035c — YLR221c — YML030w

[0578] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description andaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

[0579] Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

1 94 1 375 PRT Saccharomyces cerevisiae 1 Met Asp Ser Glu Val Ala AlaLeu Val Ile Asp Asn Gly Ser Gly Met 1 5 10 15 Cys Lys Ala Gly Phe AlaGly Asp Asp Ala Pro Arg Ala Val Phe Pro 20 25 30 Ser Ile Val Gly Arg ProArg His Gln Gly Ile Met Val Gly Met Gly 35 40 45 Gln Lys Asp Ser Tyr ValGly Asp Glu Ala Gln Ser Lys Arg Gly Ile 50 55 60 Leu Thr Leu Arg Tyr ProIle Glu His Gly Ile Val Thr Asn Trp Asp 65 70 75 80 Asp Met Glu Lys IleTrp His His Thr Phe Tyr Asn Glu Leu Arg Val 85 90 95 Ala Pro Glu Glu HisPro Val Leu Leu Thr Glu Ala Pro Met Asn Pro 100 105 110 Lys Ser Asn ArgGlu Lys Met Thr Gln Ile Met Phe Glu Thr Phe Asn 115 120 125 Val Pro AlaPhe Tyr Val Ser Ile Gln Ala Val Leu Ser Leu Tyr Ser 130 135 140 Ser GlyArg Thr Thr Gly Ile Val Leu Asp Ser Gly Asp Gly Val Thr 145 150 155 160His Val Val Pro Ile Tyr Ala Gly Phe Ser Leu Pro His Ala Ile Leu 165 170175 Arg Ile Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile 180185 190 Leu Ser Glu Arg Gly Tyr Ser Phe Ser Thr Thr Ala Glu Arg Glu Ile195 200 205 Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp PheGlu 210 215 220 Gln Glu Met Gln Thr Ala Ala Gln Ser Ser Ser Ile Glu LysSer Tyr 225 230 235 240 Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly AsnGlu Arg Phe Arg 245 250 255 Ala Pro Glu Ala Leu Phe His Pro Ser Val LeuGly Leu Glu Ser Ala 260 265 270 Gly Ile Asp Gln Thr Thr Tyr Asn Ser IleMet Lys Cys Asp Val Asp 275 280 285 Val Arg Lys Glu Leu Tyr Gly Asn IleVal Met Ser Gly Gly Thr Thr 290 295 300 Met Phe Pro Gly Ile Ala Glu ArgMet Gln Lys Glu Ile Thr Ala Leu 305 310 315 320 Ala Pro Ser Ser Met LysVal Lys Ile Ile Ala Pro Pro Glu Arg Lys 325 330 335 Tyr Ser Val Trp IleGly Gly Ser Ile Leu Ala Ser Leu Thr Thr Phe 340 345 350 Gln Gln Met TrpIle Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser 355 360 365 Ile Val HisHis Lys Cys Phe 370 375 2 1125 DNA Saccharomyces cerevisiae 2 atggattctggggttgctgc tttggttatt gataacggtt ctggtatgtg taaagccggt 60 tttgccggtgacgacgctcc tcgtgctgtc ttcccatcta tcgtcggtag accaagacac 120 caaggtatcatggtcggtat gggtcaaaaa gactcctacg ttggtgatga agctcaatcc 180 aagagaggtatcttgacttt acgttaccca attgaacacg gtattgtcac caactgggac 240 gatatggaaaagatctggca tcataccttc tacaacgaat tgagagttgc cccagaagaa 300 caccctgttcttttgactga agctccaatg aaccctaaat caaacagaga aaagatgact 360 caaattatgtttgaaacttt caacgttcca gccttctacg tttccatcca agccgttttg 420 tccttgtactcttccggtag aactactggt attgttttgg attccggtga tggtgttact 480 cacgtcgttccaatttacgc tggtttctct ctacctcacg ccattttgag aatcgatttg 540 gccggtagagatttgactga ctacttgatg aagatcttga gtgaacgtgg ttactctttc 600 tccaccactgctgaaagaga aattgtccgt gacatcaagg aaaaactatg ttacgtcgcc 660 ttggacttcgaacaagaaat gcaaaccgct gctcaatctt cttcaattga aaaatcctac 720 gaacttccagatggtcaagt catcactatt ggtaacgaaa gattcagagc cccagaagct 780 ttgttccatccttctgtttt gggtttggaa tctgccggta ttgaccaaac tacttacaac 840 tccatcatgaagtgtgatgt cgatgtccgt aaggaattat acggtaacat cgttatgtcc 900 ggtggtaccaccatgttccc aggtattgcc gaaagaatgc aaaaggaaat caccgctttg 960 gctccatcttccatgaaggt caagatcatt gctcctccag aaagaaagta ctccgtctgg 1020 attggtggttctatcttggc ttctttgact accttccaac aaatgtggat ctcaaaacaa 1080 gaatacgacgaaagtggtcc atctatcgtt caccacaagt gtttc 1125 3 1357 PRT Saccharomycescerevisiae 3 Met Asn Val Tyr Asp Asp Val Leu Asp Ala Thr Val Val Ser HisSer 1 5 10 15 Leu Ala Thr His Phe Thr Thr Ser Asp Tyr Glu Glu Leu LeuVal Val 20 25 30 Arg Thr Asn Ile Leu Ser Val Tyr Arg Pro Thr Arg Asp GlyLys Leu 35 40 45 Tyr Leu Thr Asp Glu Phe Lys Phe His Gly Leu Ile Thr AspIle Gly 50 55 60 Leu Ile Pro Gln Lys Asp Ser Pro Leu Ser Cys Leu Leu LeuCys Thr 65 70 75 80 Gly Val Ala Lys Ile Ser Ile Leu Lys Phe Asn Thr LeuThr Asn Ser 85 90 95 Ile Asp Thr Leu Ser Leu His Tyr Tyr Glu Gly Lys PheLys Gly Lys 100 105 110 Ser Leu Val Glu Leu Ala Lys Ile Ser Thr Leu ArgMet Asp Pro Gly 115 120 125 Ser Ser Cys Ala Leu Leu Phe Asn Asn Asp IleIle Ala Phe Leu Pro 130 135 140 Phe His Val Asn Lys Asn Asp Asp Asp GluGlu Glu Glu Asp Glu Asp 145 150 155 160 Glu Asn Ile Asp Asp Ser Glu LeuIle His Ser Met Asn Gln Lys Ser 165 170 175 Gln Gly Thr Asn Thr Phe AsnLys Arg Lys Arg Thr Lys Leu Gly Asp 180 185 190 Lys Phe Thr Ala Pro SerVal Val Leu Val Ala Ser Glu Leu Tyr Glu 195 200 205 Gly Ala Lys Asn IleIle Asp Ile Gln Phe Leu Lys Asn Phe Thr Lys 210 215 220 Pro Thr Ile AlaLeu Leu Tyr Gln Pro Lys Leu Val Trp Ala Gly Asn 225 230 235 240 Thr ThrIle Ser Lys Leu Pro Thr Gln Tyr Val Ile Leu Thr Leu Asn 245 250 255 IleGln Pro Ala Glu Ser Ala Thr Lys Ile Glu Ser Thr Thr Ile Ala 260 265 270Phe Val Lys Glu Leu Pro Trp Asp Leu His Thr Ile Val Pro Val Ser 275 280285 Asn Gly Ala Ile Ile Val Gly Thr Asn Glu Leu Ala Phe Leu Asp Asn 290295 300 Thr Gly Val Leu Gln Ser Thr Val Leu Leu Asn Ser Phe Ala Asp Lys305 310 315 320 Glu Leu Gln Lys Thr Lys Ile Ile Asn Asn Ser Ser Leu GluIle Met 325 330 335 Phe Arg Glu Lys Asn Thr Thr Ser Ile Trp Ile Pro SerSer Lys Ser 340 345 350 Lys Asn Gly Gly Ser Asn Asn Asp Glu Thr Leu LeuLeu Met Asp Leu 355 360 365 Lys Ser Asn Ile Tyr Tyr Ile Gln Met Glu AlaGlu Gly Arg Leu Leu 370 375 380 Ile Lys Phe Asp Ile Phe Lys Leu Pro IleVal Asn Asp Leu Leu Lys 385 390 395 400 Glu Asn Ser Asn Pro Lys Cys IleThr Arg Leu Asn Ala Thr Asn Ser 405 410 415 Asn Lys Asn Met Asp Leu PheIle Gly Phe Gly Ser Gly Asn Ala Leu 420 425 430 Val Leu Arg Leu Asn AsnLeu Lys Ser Thr Ile Glu Thr Arg Glu Ala 435 440 445 His Asn Pro Ser SerGly Thr Asn Ser Leu Met Asp Ile Asn Asp Asp 450 455 460 Asp Asp Glu GluMet Asp Asp Leu Tyr Ala Asp Glu Ala Pro Glu Asn 465 470 475 480 Gly LeuThr Thr Asn Asp Ser Lys Gly Thr Val Glu Thr Val Gln Pro 485 490 495 PheAsp Ile Glu Leu Leu Ser Ser Leu Arg Asn Val Gly Pro Ile Thr 500 505 510Ser Leu Thr Val Gly Lys Val Ser Ser Ile Asp Asp Val Val Lys Gly 515 520525 Leu Pro Asn Pro Asn Lys Asn Glu Tyr Ser Leu Val Ala Thr Ser Gly 530535 540 Asn Gly Ser Gly Ser His Leu Thr Val Ile Gln Thr Ser Val Gln Pro545 550 555 560 Glu Ile Glu Leu Ala Leu Lys Phe Ile Ser Ile Thr Gln IleTrp Asn 565 570 575 Leu Lys Ile Lys Gly Arg Asp Arg Tyr Leu Ile Thr ThrAsp Ser Thr 580 585 590 Lys Ser Arg Ser Asp Ile Tyr Glu Ser Asp Asn AsnPhe Lys Leu His 595 600 605 Lys Gly Gly Arg Leu Arg Arg Asp Ala Thr ThrVal Tyr Ile Ser Met 610 615 620 Phe Gly Glu Glu Lys Arg Ile Ile Gln ValThr Thr Asn His Leu Tyr 625 630 635 640 Leu Tyr Asp Thr His Phe Arg ArgLeu Thr Thr Ile Lys Phe Asp Tyr 645 650 655 Glu Val Ile His Val Ser ValMet Asp Pro Tyr Ile Leu Val Thr Val 660 665 670 Ser Arg Gly Asp Ile LysIle Phe Glu Leu Glu Glu Lys Asn Lys Arg 675 680 685 Lys Leu Leu Lys ValAsp Leu Pro Glu Ile Leu Asn Glu Met Val Ile 690 695 700 Thr Ser Gly LeuIle Leu Lys Ser Asn Met Cys Asn Glu Phe Leu Ile 705 710 715 720 Gly LeuSer Lys Ser Gln Glu Glu Gln Leu Leu Phe Thr Phe Val Thr 725 730 735 AlaAsp Asn Gln Ile Ile Phe Phe Thr Lys Asp His Asn Asp Arg Ile 740 745 750Phe Gln Leu Asn Gly Val Asp Gln Leu Asn Glu Ser Leu Tyr Ile Ser 755 760765 Thr Tyr Gln Leu Gly Asp Glu Ile Val Pro Asp Pro Ser Ile Lys Gln 770775 780 Val Met Ile Asn Lys Leu Gly His Asp Asn Lys Glu Glu Tyr Leu Thr785 790 795 800 Ile Leu Thr Phe Gly Gly Glu Ile Tyr Gln Tyr Arg Lys LeuPro Gln 805 810 815 Arg Arg Ser Arg Phe Tyr Arg Asn Val Thr Arg Asn AspLeu Ala Ile 820 825 830 Thr Gly Ala Pro Asp Asn Ala Tyr Ala Lys Gly ValSer Ser Ile Glu 835 840 845 Arg Ile Met His Tyr Phe Pro Asp Tyr Asn GlyTyr Ser Val Ile Phe 850 855 860 Val Thr Gly Ser Val Pro Tyr Ile Leu IleLys Glu Asp Asp Ser Thr 865 870 875 880 Pro Lys Ile Phe Lys Phe Gly AsnIle Pro Leu Val Ser Val Thr Pro 885 890 895 Trp Ser Glu Arg Ser Val MetCys Val Asp Asp Ile Lys Asn Ala Arg 900 905 910 Val Tyr Thr Leu Thr ThrAsp Asn Met Tyr Tyr Gly Asn Lys Leu Pro 915 920 925 Leu Lys Gln Ile LysIle Ser Asn Val Leu Asp Asp Tyr Lys Thr Leu 930 935 940 Gln Lys Leu ValTyr His Glu Arg Ala Gln Leu Phe Leu Val Ser Tyr 945 950 955 960 Cys LysArg Val Pro Tyr Glu Ala Leu Gly Glu Asp Gly Glu Lys Val 965 970 975 IleGly Tyr Asp Glu Asn Val Pro His Ala Glu Gly Phe Gln Ser Gly 980 985 990Ile Leu Leu Ile Asn Pro Lys Ser Trp Lys Val Ile Asp Lys Ile Asp 995 10001005 Phe Pro Lys Asn Ser Val Val Asn Glu Met Arg Ser Ser Met Ile 10101015 1020 Gln Ile Asn Ser Lys Thr Lys Arg Lys Arg Glu Tyr Ile Ile Ala1025 1030 1035 Gly Val Ala Asn Ala Thr Thr Glu Asp Thr Pro Pro Thr GlyAla 1040 1045 1050 Phe His Ile Tyr Asp Val Ile Glu Val Val Pro Glu ProGly Lys 1055 1060 1065 Pro Asp Thr Asn Tyr Lys Leu Lys Glu Ile Phe GlnGlu Glu Val 1070 1075 1080 Ser Gly Thr Val Ser Thr Val Cys Glu Val SerGly Arg Phe Met 1085 1090 1095 Ile Ser Gln Ser Gln Lys Val Leu Val ArgAsp Ile Gln Glu Asp 1100 1105 1110 Asn Ser Val Ile Pro Val Ala Phe LeuAsp Ile Pro Val Phe Val 1115 1120 1125 Thr Asp Ser Lys Ser Phe Gly AsnLeu Leu Ile Ile Gly Asp Ala 1130 1135 1140 Met Gln Gly Phe Gln Phe IleGly Phe Asp Ala Glu Pro Tyr Arg 1145 1150 1155 Met Ile Ser Leu Gly ArgSer Met Ser Lys Phe Gln Thr Met Ser 1160 1165 1170 Leu Glu Phe Leu ValAsn Gly Gly Asp Met Tyr Phe Ala Ala Thr 1175 1180 1185 Asp Ala Asp ArgAsn Val His Val Leu Lys Tyr Ala Pro Asp Glu 1190 1195 1200 Pro Asn SerLeu Ser Gly Gln Arg Leu Val His Cys Ser Ser Phe 1205 1210 1215 Thr LeuHis Ser Thr Asn Ser Cys Met Met Leu Leu Pro Arg Asn 1220 1225 1230 GluGlu Phe Gly Ser Pro Gln Val Pro Ser Phe Gln Asn Val Gly 1235 1240 1245Gly Gln Val Asp Gly Ser Val Phe Lys Ile Val Pro Leu Ser Glu 1250 12551260 Glu Lys Tyr Arg Arg Leu Tyr Val Ile Gln Gln Gln Ile Ile Asp 12651270 1275 Arg Glu Leu Gln Leu Gly Gly Leu Asn Pro Arg Met Glu Arg Leu1280 1285 1290 Ala Asn Asp Phe Tyr Gln Met Gly His Ser Met Arg Pro MetLeu 1295 1300 1305 Asp Phe Asn Val Ile Arg Arg Phe Cys Gly Leu Ala IleAsp Arg 1310 1315 1320 Arg Lys Ser Ile Ala Gln Lys Ala Gly Arg His AlaHis Phe Glu 1325 1330 1335 Ala Trp Arg Asp Ile Ile Asn Ile Glu Phe SerMet Arg Ser Leu 1340 1345 1350 Cys Gln Gly Lys 1355 4 2000 DNASaccharomyces cerevisiae 4 atgaatgtat atgatgatgt gcttgacgct actgttgtgtctcattcatt agcaacacat 60 tttactactt cagattatga ggagctctta gtggtgcgaacaaatatcct ttctgtctac 120 aggcctacta gggatggtaa actgtatttg actgatgagtttaagtttca tggcttgatc 180 accgatatag gcctcattcc ccaaaaagac agtccgttaagttgcctgct tttatgcaca 240 ggcgtcgcta aaatctctat tttaaagttc aacacgctaacaaactcaat cgatacccta 300 agcctgcatt actacgaagg aaagttcaag ggtaaatcgttggtagagct ggccaaaatt 360 tccaccttga gaatggatcc tggaagttct tgtgccttactgttcaataa tgacattatc 420 gcttttctac ccttccatgt caataaaaat gacgacgatgaggaggaaga agatgaggac 480 gagaacatag acgatagtga attaatccat agcatgaatcaaaaatccca agggacgaat 540 actttcaata agaggaagag aaccaagtta ggtgacaagttcacggctcc aagcgtggta 600 ttggtagcga gtgagttgta cgaaggcgcc aaaaatatcatagatattca gttcttgaaa 660 aattttacta aaccaacgat agcgctctta taccaaccaaagcttgtttg ggcaggaaac 720 accaccattt caaaactccc tacacaatac gttatactcacattaaatat ccagcctgca 780 gaaagtgcca ccaagattga atcaaccaca atagcatttgtcaaggaact gccttgggat 840 ctacacacca ttgtacctgt ttcgaatggt gctatcattgtgggaaccaa tgaactggca 900 tttctagata atactggcgt tttacaatcg acagtattattaaactcatt tgctgataaa 960 gagttacaga aaacaaaaat tatcaacaat tcttcattggaaattatgtt cagggagaaa 1020 aacactacgt caatttggat accttcgtcc aagagcaaaaacggaggaag taataatgac 1080 gaaacactac tacttatgga cttgaaatcc aatatatactatatccaaat ggaagcagag 1140 ggcaggttac tgattaaatt cgatattttc aagctacctatagtcaatga tcttttgaag 1200 gagaactcca atccaaaatg tataacacgt ttgaatgccaccaattcaaa caaaaatatg 1260 gacttattta ttgggtttgg ttcaggaaat gctttggttctcagattgaa taatttgaaa 1320 tctaccattg aaacaagaga agcacacaat ccatcttcaggtacaaatag tttgatggat 1380 atcaatgacg acgatgatga ggaaatggac gatttatacgccgatgaagc tcctgaaaat 1440 ggactaacaa ctaatgattc caaaggcact gtggaaactgttcaaccttt cgatatcgaa 1500 ctgttatcat ctctaaggaa tgttggtccc atcacatcgttaactgtagg taaggtatct 1560 tccattgatg atgtggtaaa aggactacca aatccaaacaaaaacgagta ttcgctagtt 1620 gccacatcgg gaaatggttc aggttctcat ttgaccgtcatacagactag tgttcaaccg 1680 gaaattgaat tagcattgaa atttatcagt ataacacaaatctggaatct gaagatcaaa 1740 ggaagagata ggtacttaat aactactgat tccacaaaatcccggagtga catatatgaa 1800 agtgacaata acttcaaact tcataagggc ggccgtttaagaagagatgc tactacagtt 1860 tacatttcaa tgtttggtga agaaaaaaga ataatacaagtcaccaccaa tcacttatat 1920 ttatatgata cacattttag acgtctcacc acaattaaatttgactacga agttattcat 1980 gtttccgtca tggatccgta 2000 5 859 PRTSaccharomyces cerevisiae 5 Met Thr Tyr Lys Tyr Asn Cys Cys Asp Asp GlySer Gly Thr Thr Val 1 5 10 15 Gly Ser Val Val Arg Phe Asp Asn Val ThrLeu Leu Ile Asp Pro Gly 20 25 30 Trp Asn Pro Ser Lys Val Ser Tyr Glu GlnCys Ile Lys Tyr Trp Glu 35 40 45 Lys Val Ile Pro Glu Ile Asp Val Ile IleLeu Ser Gln Pro Thr Ile 50 55 60 Glu Cys Leu Gly Ala His Ser Leu Leu TyrTyr Asn Phe Thr Ser His 65 70 75 80 Phe Ile Ser Arg Ile Gln Val Tyr AlaThr Leu Pro Val Ile Asn Leu 85 90 95 Gly Arg Val Ser Thr Ile Asp Ser TyrAla Ser Ala Gly Val Ile Gly 100 105 110 Pro Tyr Asp Thr Asn Lys Leu AspLeu Glu Asp Ile Glu Ile Ser Phe 115 120 125 Asp His Ile Val Pro Leu LysTyr Ser Gln Leu Val Asp Leu Arg Ser 130 135 140 Arg Tyr Asp Gly Leu ThrLeu Leu Ala Tyr Asn Ala Gly Val Cys Pro 145 150 155 160 Gly Gly Ser IleTrp Cys Ile Ser Thr Tyr Ser Glu Lys Leu Val Tyr 165 170 175 Ala Lys ArgTrp Asn His Thr Arg Asp Asn Ile Leu Asn Ala Ala Ser 180 185 190 Ile LeuAsp Ala Thr Gly Lys Pro Leu Ser Thr Leu Met Arg Pro Ser 195 200 205 AlaIle Ile Thr Thr Leu Asp Arg Phe Gly Ser Ser Gln Pro Phe Lys 210 215 220Lys Arg Ser Lys Ile Phe Lys Asp Thr Leu Lys Lys Gly Leu Ser Ser 225 230235 240 Asp Gly Ser Val Ile Ile Pro Val Asp Met Ser Gly Lys Phe Leu Asp245 250 255 Leu Phe Thr Gln Val His Glu Leu Leu Phe Glu Ser Thr Lys IleAsn 260 265 270 Ala His Thr Gln Val Pro Val Leu Ile Leu Ser Tyr Ala ArgGly Arg 275 280 285 Thr Leu Thr Tyr Ala Lys Ser Met Leu Glu Trp Leu SerPro Ser Leu 290 295 300 Leu Lys Thr Trp Glu Asn Arg Asn Asn Thr Ser ProPhe Glu Ile Gly 305 310 315 320 Ser Arg Ile Lys Ile Ile Ala Pro Asn GluLeu Ser Lys Tyr Pro Gly 325 330 335 Ser Lys Ile Cys Phe Val Ser Glu ValGly Ala Leu Ile Asn Glu Val 340 345 350 Ile Ile Lys Val Gly Asn Ser GluLys Thr Thr Leu Ile Leu Thr Lys 355 360 365 Pro Ser Phe Glu Cys Ala SerSer Leu Asp Lys Ile Leu Glu Ile Val 370 375 380 Glu Gln Asp Glu Arg AsnTrp Lys Thr Phe Pro Glu Asp Gly Lys Ser 385 390 395 400 Phe Leu Cys AspAsn Tyr Ile Ser Ile Asp Thr Ile Lys Glu Glu Pro 405 410 415 Leu Ser LysGlu Glu Thr Glu Ala Phe Lys Val Gln Leu Lys Glu Lys 420 425 430 Lys ArgAsp Arg Asn Lys Lys Ile Leu Leu Val Lys Arg Glu Ser Lys 435 440 445 LysLeu Ala Asn Gly Asn Ala Ile Ile Asp Asp Thr Asn Gly Glu Arg 450 455 460Ala Met Arg Asn Gln Asp Ile Leu Val Glu Asn Val Asn Gly Val Pro 465 470475 480 Pro Ile Asp His Ile Met Gly Gly Asp Glu Asp Asp Asp Glu Glu Glu485 490 495 Glu Asn Asp Asn Leu Leu Asn Leu Leu Lys Asp Asn Ser Glu LysSer 500 505 510 Ala Ala Lys Lys Asn Thr Glu Val Pro Val Asp Ile Ile IleGln Pro 515 520 525 Ser Ala Ala Ser Lys His Lys Met Phe Pro Phe Asn ProAla Lys Ile 530 535 540 Lys Lys Asp Asp Tyr Gly Thr Val Val Asp Phe ThrMet Phe Leu Pro 545 550 555 560 Asp Asp Ser Asp Asn Val Asn Gln Asn SerArg Lys Arg Pro Leu Lys 565 570 575 Asp Gly Ala Lys Thr Thr Ser Pro ValAsn Glu Glu Asp Asn Lys Asn 580 585 590 Glu Glu Glu Asp Gly Tyr Asn MetSer Asp Pro Ile Ser Lys Arg Ser 595 600 605 Lys His Arg Ala Ser Arg TyrSer Gly Phe Ser Gly Thr Gly Glu Ala 610 615 620 Glu Asn Phe Asp Asn LeuAsp Tyr Leu Lys Ile Asp Lys Thr Leu Ser 625 630 635 640 Lys Arg Thr IleSer Thr Val Asn Val Gln Leu Lys Cys Ser Val Val 645 650 655 Ile Leu AsnLeu Gln Ser Leu Val Asp Gln Arg Ser Ala Ser Ile Ile 660 665 670 Trp ProSer Leu Lys Ser Arg Lys Ile Val Leu Ser Ala Pro Lys Gln 675 680 685 IleGln Asn Glu Glu Ile Thr Ala Lys Leu Ile Lys Lys Asn Ile Glu 690 695 700Val Val Asn Met Pro Leu Asn Lys Ile Val Glu Phe Ser Thr Thr Ile 705 710715 720 Lys Thr Leu Asp Ile Ser Ile Asp Ser Asn Leu Asp Asn Leu Leu Lys725 730 735 Trp Gln Arg Ile Ser Asp Ser Tyr Thr Val Ala Thr Val Val GlyArg 740 745 750 Leu Val Lys Glu Ser Leu Pro Gln Val Asn Asn His Gln LysThr Ala 755 760 765 Ser Arg Ser Lys Leu Val Leu Lys Pro Leu His Gly SerSer Arg Ser 770 775 780 His Lys Thr Gly Ala Leu Ser Ile Gly Asp Val ArgLeu Ala Gln Leu 785 790 795 800 Lys Lys Leu Leu Thr Glu Lys Asn Tyr IleAla Glu Phe Lys Gly Glu 805 810 815 Gly Thr Leu Val Ile Asn Glu Lys ValAla Val Arg Lys Ile Asn Asp 820 825 830 Ala Glu Thr Ile Ile Asp Gly ThrPro Ser Glu Leu Phe Asp Thr Val 835 840 845 Lys Lys Leu Val Thr Asp MetLeu Ala Lys Ile 850 855 6 2000 DNA Saccharomyces cerevisiae 6 atgacttataaatacaattg ctgtgatgat ggatcaggaa ccaccgttgg ttcagtggta 60 cgatttgataacgtcacctt gttaatcgat cccggttgga atccatctaa agtatcgtat 120 gagcaatgcatcaagtactg ggaaaaagtg ataccggaaa ttgatgtaat aatactatca 180 caaccaacaattgaatgttt aggtgctcat tcccttctgt actataattt tacctcgcat 240 ttcatatccagaattcaagt ttacgcaact cttcctgtta tcaacttagg gagagtttcc 300 accatagattcatatgcatc tgcaggtgtt ataggtccat atgacacaaa taaactcgat 360 cttgaagatattgagatatc ttttgatcat attgtacctt tgaagtactc gcaattggta 420 gatctacgatcaagatacga tggattgact ttactggcgt acaatgctgg tgtgtgtcca 480 ggtggttctatttggtgcat tagcacatac tcagagaaat tggtttatgc aaagcgatgg 540 aaccacactagagataacat actaaatgct gcttctatat tggacgccac tggtaagccc 600 ttatcaactttgatgaggcc ctctgcaata attactactt tggacaggtt tggctcctct 660 caaccgtttaaaaaacgttc aaaaatcttt aaagatacat taaagaaagg tttgagttcg 720 gatgggtccgttataatacc ggtagatatg agtggcaaat ttctggacct gttcacacaa 780 gttcatgagttattatttga aagcacaaag atcaatgcgc acacacaagt acctgtactt 840 attctgtcttacgcgagagg aagaactcta acatatgcca aatcaatgct cgagtggctg 900 tccccttcactacttaagac atgggaaaac agaaataata cttctccatt tgaaattgga 960 tcacggatcaaaatcatcgc accaaacgaa ttgagtaaat atcctggttc aaagatttgc 1020 ttcgtatctgaggtaggagc tttgattaat gaagtgataa ttaaagttgg taattctgag 1080 aagacaaccttgattttgac caagccgagc tttgaatgtg cgtcgtcttt agacaagata 1140 ctagaaattgttgaacaaga tgaaagaaat tggaaaacat ttcctgaaga tggtaaatca 1200 ttcctctgcgataattacat ttcaatagac accataaaag aagaaccctt gagtaaagag 1260 gaaacagaggcgtttaaagt acagcttaaa gaaaagaaaa gggacagaaa taagaaaatt 1320 ctactggtgaaaagagagtc taagaaactg gctaacggca acgcaatcat agatgatacc 1380 aacggagagagggcaatgag aaatcaagac attttagtgg aaaacgtcaa tggagtacca 1440 ccgattgatcatattatggg gggtgacgaa gatgatgatg aagaggaaga aaacgacaat 1500 ttgttgaatttactcaagga taactctgag aaatcagcag cgaaaaaaaa tacggaagtt 1560 cctgtagacattataattca accaagtgcc gcttcaaaac ataaaatgtt cccatttaac 1620 cccgctaaaatcaagaaaga tgattatggt actgttgtag attttacgat gtttttacct 1680 gatgattctgacaatgttaa tcaaaatagc aggaaaagac ctctcaaaga cggtgctaaa 1740 accacgagccctgtaaacga agaagataac aaaaatgaag aagaagatgg ttataatatg 1800 agtgatccaattagcaaaag gagcaaacac cgtgcttctc gttactcggg attttctgga 1860 accggagaagcagaaaattt tgataacctt gactatttaa agatagacaa aacgctctcc 1920 aaaagaacaatctcaactgt aaatgtccaa ttgaaatgct ccgtagtaat cctaaattta 1980 cagagtttagttgatcaaag 2000 7 372 PRT Saccharomyces cerevisiae 7 Met Lys Cys Arg ValTrp Ser Glu Ala Arg Val Tyr Thr Asn Ile Asn 1 5 10 15 Lys Gln Arg ThrGlu Glu Tyr Trp Asp Tyr Glu Asn Thr Val Ile Asp 20 25 30 Trp Ser Thr AsnThr Lys Asp Tyr Glu Ile Glu Asn Lys Val Gly Arg 35 40 45 Gly Lys Tyr SerGlu Val Phe Gln Gly Val Lys Leu Asp Ser Lys Val 50 55 60 Lys Ile Val IleLys Met Leu Lys Pro Val Lys Lys Lys Lys Ile Lys 65 70 75 80 Arg Glu IleLys Ile Leu Thr Asp Leu Ser Asn Glu Lys Val Pro Pro 85 90 95 Thr Thr LeuPro Phe Gln Lys Asp Gln Tyr Tyr Thr Asn Gln Lys Glu 100 105 110 Asp ValLeu Lys Phe Ile Arg Pro Tyr Ile Phe Asp Gln Pro His Asn 115 120 125 GlyHis Ala Asn Ile Ile His Leu Phe Asp Ile Ile Lys Asp Pro Ile 130 135 140Ser Lys Thr Pro Ala Leu Val Phe Glu Tyr Val Asp Asn Val Asp Phe 145 150155 160 Arg Ile Leu Tyr Pro Lys Leu Thr Asp Leu Glu Ile Arg Phe Tyr Met165 170 175 Phe Glu Leu Leu Lys Ala Leu Asp Tyr Cys His Ser Met Gly IleMet 180 185 190 His Arg Asp Val Lys Pro His Asn Val Met Ile Asp His LysAsn Lys 195 200 205 Lys Leu Arg Leu Ile Asp Trp Gly Leu Ala Glu Phe TyrHis Val Asn 210 215 220 Met Glu Tyr Asn Val Arg Val Ala Ser Arg Phe PheLys Gly Pro Glu 225 230 235 240 Leu Leu Val Asp Tyr Arg Met Tyr Asp TyrSer Leu Asp Leu Trp Ser 245 250 255 Phe Gly Thr Met Leu Ala Ser Met IlePhe Lys Arg Glu Pro Phe Phe 260 265 270 His Gly Thr Ser Asn Thr Asp GlnLeu Val Lys Ile Val Lys Val Leu 275 280 285 Gly Thr Ser Asp Phe Glu LysTyr Leu Leu Lys Tyr Glu Ile Thr Leu 290 295 300 Pro Arg Glu Phe Tyr AspMet Asp Gln Tyr Ile Arg Lys Pro Trp His 305 310 315 320 Arg Phe Ile AsnAsp Gly Asn Lys His Leu Ser Gly Asn Asp Glu Ile 325 330 335 Ile Asp LeuIle Asp Asn Leu Leu Arg Tyr Asp His Gln Glu Arg Leu 340 345 350 Thr AlaLys Glu Ala Met Gly His Pro Trp Phe Ala Pro Ile Arg Glu 355 360 365 GlnIle Glu Lys 370 8 1119 DNA Saccharomyces cerevisiae 8 atgaaatgcagggtatggtc agaggctcgt gtttatacga atatcaataa acaaagaacc 60 gaggaatattgggattatga aaatactgta attgattggt ccacaaatac aaaggactat 120 gaaattgaaaataaagttgg gcgaggaaaa tactccgagg ttttccaagg tgtcaaatta 180 gactctaaagttaaaattgt tattaagatg ttgaaaccag ttaaaaagaa gaagatcaag 240 agagaaatcaaaatcttaac ggatttgtct aacgaaaaag tgcctccaac gactttgcca 300 tttcaaaaagatcaatatta cacaaatcaa aaggaggacg ttttaaaatt catcaggccc 360 tatatctttgatcagccaca caatggtcat gcaaacataa ttcatctatt tgatataata 420 aaggatcccatctcaaaaac tccggctttg gtcttcgaat acgtagataa tgtggacttc 480 cgtattctttaccctaaatt aaccgatctc gaaattaggt tttatatgtt tgagttatta 540 aaggccttagactattgtca ttcaatggga ataatgcata gagatgttaa acctcataac 600 gtaatgattgatcataagaa taaaaaattg cgattgatag attgggggct tgctgaattt 660 tatcatgttaatatggaata caatgttcgt gttgcgtcga ggttttttaa gggtcctgaa 720 ctactagttgactacagaat gtatgattat tctttagact tgtggtcgtt tgggacaatg 780 ttggcttctatgatctttaa aagagagcca tttttccatg gaacgagtaa cacagaccag 840 cttgtcaagatcgtcaaagt acttggtaca agcgattttg agaaatacct gttgaagtat 900 gaaattaccttaccgagaga attttacgat atggaccaat acatcagaaa gccttggcat 960 agattcatcaatgatggtaa taaacattta agcggcaacg atgaaattat tgaccttatt 1020 gacaatcttttgagatatga tcatcaagaa agattaactg ctaaggaagc gatgggacac 1080 ccgtggtttgccccaataag ggaacaaatt gaaaaataa 1119 9 445 PRT Saccharomyces cerevisiae9 Met Ala Ser Leu Pro Gly Ile Asp Glu His Thr Thr Ser Glu Glu Leu 1 5 1015 Ile Thr Gly Asp Asn Glu Trp His Lys Leu Val Ile Pro Lys Gly Ser 20 2530 Asp Trp Gln Ile Asp Leu Lys Ala Glu Gly Lys Leu Ile Val Lys Val 35 4045 Asn Ser Gly Ile Val Glu Ile Phe Gly Thr Glu Leu Ala Val Asp Asp 50 5560 Glu Tyr Thr Phe Gln Asn Trp Lys Phe Pro Ile Tyr Ala Val Glu Glu 65 7075 80 Thr Glu Leu Leu Trp Lys Cys Pro Asp Leu Thr Thr Asn Thr Ile Thr 8590 95 Val Lys Pro Asn His Thr Met Lys Tyr Ile Tyr Asn Leu His Phe Met100 105 110 Leu Glu Lys Ile Arg Met Ser Asn Phe Glu Gly Pro Arg Val ValIle 115 120 125 Val Gly Gly Ser Gln Thr Gly Lys Thr Ser Leu Ser Arg ThrLeu Cys 130 135 140 Ser Tyr Ala Leu Lys Phe Asn Ala Tyr Gln Pro Leu TyrIle Asn Leu 145 150 155 160 Asp Pro Gln Gln Pro Ile Phe Thr Val Pro GlyCys Ile Ser Ala Thr 165 170 175 Pro Ile Ser Asp Ile Leu Asp Ala Gln LeuPro Thr Trp Gly Gln Ser 180 185 190 Leu Thr Ser Gly Ala Thr Leu Leu HisAsn Lys Gln Pro Met Val Lys 195 200 205 Asn Phe Gly Leu Glu Arg Ile AsnGlu Asn Lys Asp Leu Tyr Leu Glu 210 215 220 Cys Ile Ser Gln Leu Gly GlnVal Val Gly Gln Arg Leu His Leu Asp 225 230 235 240 Pro Gln Val Arg ArgSer Gly Cys Ile Val Asp Thr Pro Ser Ile Ser 245 250 255 Gln Leu Asp GluAsn Leu Ala Glu Leu His His Ile Ile Glu Lys Leu 260 265 270 Asn Val AsnIle Met Leu Val Leu Cys Ser Glu Thr Asp Pro Leu Trp 275 280 285 Glu LysVal Lys Lys Thr Phe Gly Pro Glu Leu Gly Asn Asn Asn Ile 290 295 300 PhePhe Ile Pro Lys Leu Asp Gly Val Ser Ala Val Asp Asp Val Tyr 305 310 315320 Lys Arg Ser Leu Gln Arg Thr Ser Ile Arg Glu Tyr Phe Tyr Gly Ser 325330 335 Leu Asp Thr Ala Leu Ser Pro Tyr Ala Ile Gly Val Asp Tyr Glu Asp340 345 350 Leu Thr Ile Trp Lys Pro Ser Asn Val Phe Asp Asn Glu Val GlyArg 355 360 365 Val Glu Leu Phe Pro Val Thr Ile Thr Pro Ser Asn Leu GlnHis Ala 370 375 380 Ile Ile Ala Ile Thr Phe Ala Glu Arg Arg Ala Asp GlnAla Thr Val 385 390 395 400 Ile Lys Ser Pro Ile Leu Gly Phe Ala Leu IleThr Glu Val Asn Glu 405 410 415 Lys Arg Arg Lys Leu Arg Val Leu Leu ProVal Pro Gly Arg Leu Pro 420 425 430 Ser Lys Ala Met Ile Leu Thr Ser TyrArg Tyr Leu Glu 435 440 445 10 1338 DNA Saccharomyces cerevisiae 10atggcaagtc tacctggtat tgatgagcat actacctctg aagaattgat aactggtgat 60aacgaatggc acaagttggt tattcccaag ggcagtgatt ggcaaattga tcttaaagca 120gagggaaaac tgatagtcaa ggtaaactcg ggcatagttg agatatttgg caccgagttg 180gcagtagatg atgagtacac ttttcagaac tggaagtttc ccatatacgc tgtcgaagaa 240acagaattat tatggaaatg tcctgattta actacgaata caataactgt caagcctaac 300catactatga aatatattta taatctacac ttcatgttgg agaagatacg aatgtctaac 360tttgaaggcc ctcgagtggt gattgttggt ggttcgcaaa ccgggaagac ttccctatcg 420aggacactat gttcttacgc tttgaaattc aacgcttacc aaccactata catcaatctt 480gaccctcaac agcccatttt tactgttcct ggatgcatat ctgccacccc aatatcagat 540atacttgatg cacaactacc cacttggggt cagagtctca ctagtggtgc cacactacta 600cataataagc agccaatggt gaaaaatttt ggcctggaaa ggattaatga gaacaaagat 660ctataccttg agtgtataag ccagttaggt caagtagtag gtcaaaggtt acatttggat 720cctcaagtca ggagatcagg gtgcattgtc gatacgccat caatatcaca actggatgaa 780aatttggctg aactgcacca tatcattgag aaactcaatg ttaacattat gctagtacta 840tgttctgaaa cggatcctct ttgggagaaa gtaaaaaaga catttggtcc agaattggga 900aataataata tttttttcat tcctaaattg gatggtgtat ccgcagttga tgatgtatac 960aaaagatcct tgcagaggac atccataaga gagtactttt acggctctct tgatacagcc 1020ttgagtcctt atgctattgg tgttgattat gaggatttaa ctatttggaa acctagcaat 1080gttttcgata acgaggtcgg tagggtggaa ctgttccccg tcactataac tccaagtaac 1140ctacagcacg ctattatagc cataacgttc gcagaaagaa gggcagatca ggcaacagta 1200ataaaatcgc ctattttagg attcgctttg attacagaag ttaatgaaaa aaggcgtaaa 1260ttaagggttt tactacctgt cccaggccga cttcccagca aggcgatgat tctaacttca 1320tatagatatt tagagtaa 1338 11 842 PRT Saccharomyces cerevisiae 11 Met ValAla Phe Thr Val Asp Gln Met Arg Ser Leu Met Asp Lys Val 1 5 10 15 ThrAsn Val Arg Asn Met Ser Val Ile Ala His Val Asp His Gly Lys 20 25 30 SerThr Leu Thr Asp Ser Leu Val Gln Arg Ala Gly Ile Ile Ser Ala 35 40 45 AlaLys Ala Gly Glu Ala Arg Phe Thr Asp Thr Arg Lys Asp Glu Gln 50 55 60 GluArg Gly Ile Thr Ile Lys Ser Thr Ala Ile Ser Leu Tyr Ser Glu 65 70 75 80Met Ser Asp Glu Asp Val Lys Glu Ile Lys Gln Lys Thr Asp Gly Asn 85 90 95Ser Phe Leu Ile Asn Leu Ile Asp Ser Pro Gly His Val Asp Phe Ser 100 105110 Ser Glu Val Thr Ala Ala Leu Arg Val Thr Asp Gly Ala Leu Val Val 115120 125 Val Asp Thr Ile Glu Gly Val Cys Val Gln Thr Glu Thr Val Leu Arg130 135 140 Gln Ala Leu Gly Glu Arg Ile Lys Pro Val Val Val Ile Asn LysVal 145 150 155 160 Asp Arg Ala Leu Leu Glu Leu Gln Val Ser Lys Glu AspLeu Tyr Gln 165 170 175 Thr Phe Ala Arg Thr Val Glu Ser Val Asn Val IleVal Ser Thr Tyr 180 185 190 Ala Asp Glu Val Leu Gly Asp Val Gln Val TyrPro Ala Arg Gly Thr 195 200 205 Val Ala Phe Gly Ser Gly Leu His Gly TrpAla Phe Thr Ile Arg Gln 210 215 220 Phe Ala Thr Arg Tyr Ala Lys Lys PheGly Val Asp Lys Ala Lys Met 225 230 235 240 Met Asp Arg Leu Trp Gly AspSer Phe Phe Asn Pro Lys Thr Lys Lys 245 250 255 Trp Thr Asn Lys Asp ThrAsp Ala Glu Gly Lys Pro Leu Glu Arg Ala 260 265 270 Phe Asn Met Phe IleLeu Asp Pro Ile Phe Arg Leu Phe Thr Ala Ile 275 280 285 Met Asn Phe LysLys Asp Glu Ile Pro Val Leu Leu Glu Lys Leu Glu 290 295 300 Ile Val LeuLys Gly Asp Glu Lys Asp Leu Glu Gly Lys Ala Leu Leu 305 310 315 320 LysVal Val Met Arg Lys Phe Leu Pro Ala Ala Asp Ala Leu Leu Glu 325 330 335Met Ile Val Leu His Leu Pro Ser Pro Val Thr Ala Gln Ala Tyr Arg 340 345350 Ala Glu Gln Leu Tyr Glu Gly Pro Ala Asp Asp Ala Asn Cys Ile Ala 355360 365 Ile Lys Asn Cys Asp Pro Lys Ala Asp Leu Met Leu Tyr Val Ser Lys370 375 380 Met Val Pro Thr Ser Asp Lys Gly Arg Phe Tyr Ala Phe Gly ArgVal 385 390 395 400 Phe Ala Gly Thr Val Lys Ser Gly Gln Lys Val Arg IleGln Gly Pro 405 410 415 Asn Tyr Val Pro Gly Lys Lys Asp Asp Leu Phe IleLys Ala Ile Gln 420 425 430 Arg Val Val Leu Met Met Gly Arg Phe Val GluPro Ile Asp Asp Cys 435 440 445 Pro Ala Gly Asn Ile Ile Gly Leu Val GlyIle Asp Gln Phe Leu Leu 450 455 460 Lys Thr Gly Thr Leu Thr Thr Ser GluThr Ala His Asn Met Lys Val 465 470 475 480 Met Lys Phe Ser Val Ser ProVal Val Gln Val Ala Val Glu Val Lys 485 490 495 Asn Ala Asn Asp Leu ProLys Leu Val Glu Gly Leu Lys Arg Leu Ser 500 505 510 Lys Ser Asp Pro CysVal Leu Thr Tyr Met Ser Glu Ser Gly Glu His 515 520 525 Ile Val Ala GlyThr Gly Glu Leu His Leu Glu Ile Cys Leu Gln Asp 530 535 540 Leu Glu HisAsp His Ala Gly Val Pro Leu Lys Ile Ser Pro Pro Val 545 550 555 560 ValAla Tyr Arg Glu Thr Val Glu Ser Glu Ser Ser Gln Thr Ala Leu 565 570 575Ser Lys Ser Pro Asn Lys His Asn Arg Ile Tyr Leu Lys Ala Glu Pro 580 585590 Ile Asp Glu Glu Val Ser Leu Ala Ile Glu Asn Gly Ile Ile Asn Pro 595600 605 Arg Asp Asp Phe Lys Ala Arg Ala Arg Ile Met Ala Asp Asp Tyr Gly610 615 620 Trp Asp Val Thr Asp Ala Arg Lys Ile Trp Cys Phe Gly Pro AspGly 625 630 635 640 Asn Gly Pro Asn Leu Val Ile Asp Gln Thr Lys Ala ValGln Tyr Leu 645 650 655 His Glu Ile Lys Asp Ser Val Val Ala Ala Phe GlnTrp Ala Thr Lys 660 665 670 Glu Gly Pro Ile Phe Gly Glu Glu Met Arg SerVal Arg Val Asn Ile 675 680 685 Leu Asp Val Thr Leu His Ala Asp Ala IleHis Arg Gly Gly Gly Gln 690 695 700 Ile Ile Pro Thr Met Arg Arg Ala ThrTyr Ala Gly Phe Leu Leu Ala 705 710 715 720 Asp Pro Lys Ile Gln Glu ProVal Phe Leu Val Glu Ile Gln Cys Pro 725 730 735 Glu Gln Ala Val Gly GlyIle Tyr Ser Val Leu Asn Lys Lys Arg Gly 740 745 750 Gln Val Val Ser GluGlu Gln Arg Pro Gly Thr Pro Leu Phe Thr Val 755 760 765 Lys Ala Tyr LeuPro Val Asn Glu Ser Phe Gly Phe Thr Gly Glu Leu 770 775 780 Arg Gln AlaThr Gly Gly Gln Ala Phe Pro Gln Met Val Phe Asp His 785 790 795 800 TrpSer Thr Leu Gly Ser Asp Pro Leu Asp Pro Thr Ser Lys Ala Gly 805 810 815Glu Ile Val Leu Ala Ala Arg Lys Arg His Gly Met Lys Glu Glu Val 820 825830 Pro Gly Trp Gln Glu Tyr Tyr Asp Lys Leu 835 840 12 2529 DNASaccharomyces cerevisiae 12 atggttgctt tcactgttga ccaaatgcgt tctttaatggacaaagttac caatgtgcgt 60 aacatgtccg ttattgctca cgtcgatcat ggtaagtccactttgaccga ttccttggtc 120 caaagagccg gtattatttc cgctgctaag gctggtgaagctcgtttcac cgataccaga 180 aaggatgaac aagaaagagg tatcactatc aagtctaccgctatttctct atactctgaa 240 atgtctgacg aagatgtcaa ggaaatcaag caaaagaccgacggtaactc cttcttgatc 300 aacttgatcg actctccagg tcacgttgac ttctcctctgaagttactgc cgctttacgt 360 gtcactgacg gtgctttggt tgtcgtcgac accattgaaggtgtctgtgt ccaaaccgaa 420 actgttttga gacaagcttt gggtgagaga atcaagcctgttgttgttat caacaaggtc 480 gacagagctt tgttggaatt gcaagtttct aaggaagatttataccaaac ctttgccaga 540 actgttgaat ccgttaacgt catcgtttcc acctacgccgatgaagtttt gggtgatgtc 600 caagtttacc cagccagagg taccgttgcc ttcggttccggtttgcacgg ttgggctttc 660 actatccgtc aattcgccac cagatatgct aagaaattcggtgtcgacaa ggccaagatg 720 atggacagat tatggggtga ctctttcttc aacccaaagaccaagaagtg gaccaacaag 780 gacactgatg ctgaaggtaa gccattggaa agagctttcaacatgttcat cttggaccca 840 atcttcagat tattcactgc tatcatgaac ttcaagaaagatgaaattcc agttttgcta 900 gaaaagttgg aaattgtctt gaagggtgac gaaaaggacttggaaggtaa ggccttgttg 960 aaggttgtta tgagaaagtt cttgccagct gccgatgccttattggaaat gattgtcttg 1020 cacttgccat ctccagtcac tgctcaagcc tacagagctgaacaattata cgaaggtcca 1080 gctgacgatg ccaactgtat tgctatcaag aactgtgatccaaaggctga tttgatgttg 1140 tacgtctcca agatggtgcc aacctctgat aagggtagattctacgcctt cggtagagtt 1200 tttgccggta ctgttaagtc cggtcaaaag gtcagaatccaaggtccaaa ctacgttcca 1260 ggtaagaagg acgatttgtt catcaaggcc attcaaagagttgttttgat gatgggtaga 1320 tttgtcgaac caatcgatga ctgtccagcc ggtaacattatcggtttagt cggtatcgat 1380 caattcttgt tgaagactgg tactttgacc accagtgaaactgctcacaa catgaaggtc 1440 atgaaattct ctgtctctcc agttgtgcaa gtcgctgtcgaagtcaagaa cgctaacgac 1500 ttaccaaaat tggtcgaagg tttgaagaga ttgtccaagtctgatccatg tgtcttgacc 1560 tatatgtctg aatccggtga acatatcgtt gctggtaccggtgaattgca tttggaaatt 1620 tgtttgcaag atttggaaca cgaccacgct ggtgttccattgaagatctc cccaccagtt 1680 gtcgcttaca gagaaactgt tgaaagtgaa tcttctcaaactgctttgtc caagtctcca 1740 aacaagcata acagaatcta cttgaaggct gaaccaattgacgaagaagt ctctttggct 1800 attgaaaacg gtatcatcaa cccaagagat gatttcaaggccagagctag aatcatggct 1860 gacgactacg gttgggatgt caccgatgcc agaaagatctggtgtttcgg tccagacggt 1920 aacggtccaa acttggttat tgaccaaact aaggctgtccaatacttgca cgaaatcaag 1980 gattccgttg ttgctgcttt ccaatgggct accaaggaaggtccaatttt cggtgaagaa 2040 atgagatctg tcagagttaa cattttggat gttactttacatgccgatgc tatccacaga 2100 ggtggtggtc aaatcatccc aaccatgaga agagctacttacgctggttt cttgttggct 2160 gatccaaaga tccaagaacc agttttcttg gtcgaaattcaatgtccaga acaagccgtc 2220 ggtggtatct actccgtctt aaacaagaag agaggtcaagtcgtttctga agaacaaaga 2280 ccaggtactc cattgtttac cgtcaaggcc tacttgccagttaacgaatc tttcggtttc 2340 actggtgaat tgagacaagc tactggtggt caagctttcccacaaatggt tttcgaccat 2400 tggtccactt taggttctga cccattggac ccaacctctaaggctggtga aattgttctt 2460 gctgctcgta agagacacgg tatgaaggaa gaagttccaggctggcaaga atattacgac 2520 aaattgtaa 2529 13 437 PRT Saccharomycescerevisiae 13 Met Ala Val Ser Lys Val Tyr Ala Arg Ser Val Tyr Asp SerArg Gly 1 5 10 15 Asn Pro Thr Val Glu Val Glu Leu Thr Thr Glu Lys GlyVal Phe Arg 20 25 30 Ser Ile Val Pro Ser Gly Ala Ser Thr Gly Val His GluAla Leu Glu 35 40 45 Met Arg Asp Glu Asp Lys Ser Lys Trp Met Gly Lys GlyVal Met Asn 50 55 60 Ala Val Asn Asn Val Asn Asn Val Ile Ala Ala Ala PheVal Lys Ala 65 70 75 80 Asn Leu Asp Val Lys Asp Gln Lys Ala Val Asp AspPhe Leu Leu Ser 85 90 95 Leu Asp Gly Thr Ala Asn Lys Ser Lys Leu Gly AlaAsn Ala Ile Leu 100 105 110 Gly Val Ser Met Ala Ala Ala Arg Ala Ala AlaAla Glu Lys Asn Val 115 120 125 Pro Leu Tyr Gln His Leu Ala Asp Leu SerLys Ser Lys Thr Ser Pro 130 135 140 Tyr Val Leu Pro Val Pro Phe Leu AsnVal Leu Asn Gly Gly Ser His 145 150 155 160 Ala Gly Gly Ala Leu Ala LeuGln Glu Phe Met Ile Ala Pro Thr Gly 165 170 175 Ala Lys Thr Phe Ala GluAla Met Arg Ile Gly Ser Glu Val Tyr His 180 185 190 Asn Leu Lys Ser LeuThr Lys Lys Arg Tyr Gly Ala Ser Ala Gly Asn 195 200 205 Val Gly Asp GluGly Gly Val Ala Pro Asn Ile Gln Thr Ala Glu Glu 210 215 220 Ala Leu AspLeu Ile Val Asp Ala Ile Lys Ala Ala Gly His Asp Gly 225 230 235 240 LysVal Lys Ile Gly Leu Asp Cys Ala Ser Ser Glu Phe Phe Lys Asp 245 250 255Gly Lys Tyr Asp Leu Asp Phe Lys Asn Pro Glu Ser Asp Lys Ser Lys 260 265270 Trp Leu Thr Gly Val Glu Leu Ala Asp Met Tyr His Ser Leu Met Lys 275280 285 Arg Tyr Pro Ile Val Ser Ile Glu Asp Pro Phe Ala Glu Asp Asp Trp290 295 300 Glu Ala Trp Ser His Phe Phe Lys Thr Ala Gly Ile Gln Ile ValAla 305 310 315 320 Asp Asp Leu Thr Val Thr Asn Pro Ala Arg Ile Ala ThrAla Ile Glu 325 330 335 Lys Lys Ala Ala Asp Ala Leu Leu Leu Lys Val AsnGln Ile Gly Thr 340 345 350 Leu Ser Glu Ser Ile Lys Ala Ala Gln Asp SerPhe Ala Ala Asn Trp 355 360 365 Gly Val Met Val Ser His Arg Ser Gly GluThr Glu Asp Thr Phe Ile 370 375 380 Ala Asp Leu Val Val Gly Leu Arg ThrGly Gln Ile Lys Thr Gly Ala 385 390 395 400 Pro Ala Arg Ser Glu Arg LeuAla Lys Leu Asn Gln Leu Leu Arg Ile 405 410 415 Glu Glu Glu Leu Gly AspLys Ala Val Tyr Ala Gly Glu Asn Phe His 420 425 430 His Gly Asp Lys Leu435 14 1314 DNA Saccharomyces cerevisiae 14 atggctgtct ctaaagtttacgctagatcc gtctacgact cccgtggtaa cccaaccgtc 60 gaagtcgaat taaccaccgaaaagggtgtt ttcagatcca ttgttccatc tggtgcctcc 120 accggtgtcc acgaagctttggaaatgaga gatgaagaca aatccaagtg gatgggtaag 180 ggtgttatga acgctgtcaacaacgtcaac aacgtcattg ctgctgcttt cgtcaaggcc 240 aacctagatg ttaaggaccaaaaggccgtc gatgacttct tgttgtcttt ggatggtacc 300 gccaacaagt ccaagttgggtgctaacgct atcttgggtg tctccatggc cgctgctaga 360 gccgctgctg ctgaaaagaacgtcccattg taccaacatt tggctgactt gtctaagtcc 420 aagacctctc catacgttttgccagttcca ttcttgaacg ttttgaacgg tggttcccac 480 gctggtggtg ctttggctttgcaagaattc atgattgctc caactggtgc taagaccttc 540 gctgaagcca tgagaattggttccgaagtt taccacaact tgaagtcttt gaccaagaag 600 agatacggtg cttctgccggtaacgtcggt gacgaaggtg gtgttgctcc aaacattcaa 660 accgctgaag aagctttggacttgattgtt gacgctatca aggctgctgg tcacgacggt 720 aaggtcaaga tcggtttggactgtgcttcc tctgaattct tcaaggacgg taagtacgac 780 ttggacttca agaacccagaatctgacaaa tccaagtggt tgactggtgt cgaattagct 840 gacatgtacc actccttgatgaagagatac ccaattgtct ccatcgaaga tccatttgct 900 gaagatgact gggaagcttggtctcacttc ttcaagaccg ctggtatcca aattgttgct 960 gatgacttga ctgtcaccaacccagctaga attgctaccg ccatcgaaaa gaaggctgct 1020 gacgctttgt tgttgaaggttaaccaaatc ggtaccttgt ctgaatccat caaggctgct 1080 caagactctt tcgctgccaactggggtgtt atggtttccc acagatctgg tgaaactgaa 1140 gacactttca ttgctgacttggttgtcggt ttgagaactg gtcaaatcaa gactggtgct 1200 ccagctagat ccgaaagattggctaagttg aaccaattgt tgagaatcga agaagaattg 1260 ggtgacaagg ctgtctacgccggtgaaaac ttccaccacg gtgacaagtt gtaa 1314 15 312 PRT Saccharomycescerevisiae 15 Met Asp Ser Gln Pro Val Asp Val Asp Asn Ile Ile Asp ArgLeu Leu 1 5 10 15 Glu Val Arg Gly Ser Lys Pro Gly Gln Gln Val Asp LeuGlu Glu Asn 20 25 30 Glu Ile Arg Tyr Leu Cys Ser Lys Ala Arg Ser Ile PheIle Lys Gln 35 40 45 Pro Ile Leu Leu Glu Leu Glu Ala Pro Ile Lys Ile CysGly Asp Ile 50 55 60 His Gly Gln Tyr Tyr Asp Leu Leu Arg Leu Phe Glu TyrGly Gly Phe 65 70 75 80 Pro Pro Glu Ser Asn Tyr Leu Phe Leu Gly Asp TyrVal Asp Arg Gly 85 90 95 Lys Gln Ser Leu Glu Thr Ile Cys Leu Leu Leu AlaTyr Lys Ile Lys 100 105 110 Tyr Pro Glu Asn Phe Phe Ile Leu Arg Gly AsnHis Glu Cys Ala Ser 115 120 125 Ile Asn Arg Ile Tyr Gly Phe Tyr Asp GluCys Lys Arg Arg Tyr Asn 130 135 140 Ile Lys Leu Trp Lys Thr Phe Thr AspCys Phe Asn Cys Leu Pro Ile 145 150 155 160 Ala Ala Ile Ile Asp Glu LysIle Phe Cys Met His Gly Gly Leu Ser 165 170 175 Pro Asp Leu Asn Ser MetGlu Gln Ile Arg Arg Val Met Arg Pro Thr 180 185 190 Asp Ile Pro Asp ValGly Leu Leu Cys Asp Leu Leu Trp Ser Asp Pro 195 200 205 Asp Lys Asp IleVal Gly Trp Ser Glu Asn Asp Arg Gly Val Ser Phe 210 215 220 Thr Phe GlyPro Asp Val Val Asn Arg Phe Leu Gln Lys Gln Asp Met 225 230 235 240 GluLeu Ile Cys Arg Ala His Gln Val Val Glu Asp Gly Tyr Glu Phe 245 250 255Phe Ser Lys Arg Gln Leu Val Thr Leu Phe Ser Ala Pro Asn Tyr Cys 260 265270 Gly Glu Phe Asp Asn Ala Gly Ala Met Met Ser Val Asp Glu Ser Leu 275280 285 Leu Cys Ser Phe Gln Ile Leu Lys Pro Ala Gln Lys Ser Leu Pro Arg290 295 300 Gln Ala Gly Gly Arg Lys Lys Lys 305 310 16 939 DNASaccharomyces cerevisiae 16 atggactcac aaccagttga cgttgataat atcatcgatagattattgga agtaagagga 60 tctaaacctg gtcaacaagt tgatctagaa gaaaatgaaatcagatactt atgttcgaaa 120 gccagatcta tattcataaa gcaacccatt ttactagagttagaagcccc aattaaaata 180 tgtggtgaca ttcatgggca atactatgat ttactacgtctatttgagta cggtggattc 240 ccgccagaat ctaattatct atttttgggt gattatgtcgaccgtggtaa acaatcctta 300 gagactattt gtctattact ggcttacaaa attaagtatccagaaaactt tttcatttta 360 agagggaacc atgaatgtgc ttccattaat agaatttacgggttttatga tgaatgtaag 420 agacgttata atatcaaact ttggaaaact ttcacggattgtttcaattg tttaccaatt 480 gctgcaatta ttgatgagaa aatcttctgt atgcatggtggtctctcacc agatttgaat 540 agtatggaac agatcagaag ggtgatgagg ccaacagatattcccgacgt tggcttatta 600 tgtgacttat tgtggtcaga tccagataaa gatatcgtaggttggagtga aaatgataga 660 ggtgtttctt tcacttttgg tcctgatgta gtgaacagatttttacagaa acaagatatg 720 gagttgattt gcagggccca tcaagttgtg gaagatggttatgaattctt tagtaaaaga 780 caattggtga cacttttcag tgctccgaat tattgtggtgaatttgataa cgctggtgca 840 atgatgagtg ttgatgaaag tttattatgt tcttttcaaattttaaagcc agcccaaaaa 900 agtctaccaa ggcaagctgg gggtagaaag aaaaaataa 93917 247 PRT Saccharomyces cerevisiae 17 Met Pro Lys Leu Val Leu Val ArgHis Gly Gln Ser Glu Trp Asn Glu 1 5 10 15 Lys Asn Leu Phe Thr Gly TrpVal Asp Val Lys Leu Ser Ala Lys Gly 20 25 30 Gln Gln Glu Ala Ala Arg AlaGly Glu Leu Leu Lys Glu Lys Lys Val 35 40 45 Tyr Pro Asp Val Leu Tyr ThrSer Lys Leu Ser Arg Ala Ile Gln Thr 50 55 60 Ala Asn Ile Ala Leu Glu LysAla Asp Arg Leu Trp Ile Pro Val Asn 65 70 75 80 Arg Ser Trp Arg Leu AsnGlu Arg His Tyr Gly Asp Leu Gln Gly Lys 85 90 95 Asp Lys Ala Glu Thr LeuLys Lys Phe Gly Glu Glu Lys Phe Asn Thr 100 105 110 Tyr Arg Arg Ser PheAsp Val Pro Pro Pro Pro Ile Asp Ala Ser Ser 115 120 125 Pro Phe Ser GlnLys Gly Asp Glu Arg Tyr Lys Tyr Val Asp Pro Asn 130 135 140 Val Leu ProGlu Thr Glu Ser Leu Ala Leu Val Ile Asp Arg Leu Leu 145 150 155 160 ProTyr Trp Gln Asp Val Ile Ala Lys Asp Leu Leu Ser Gly Lys Thr 165 170 175Val Met Ile Ala Ala His Gly Asn Ser Leu Arg Gly Leu Val Lys His 180 185190 Leu Glu Gly Ile Ser Asp Ala Asp Ile Ala Lys Leu Asn Ile Pro Thr 195200 205 Gly Ile Pro Leu Val Phe Glu Leu Asp Glu Asn Leu Lys Pro Ser Lys210 215 220 Pro Ser Tyr Tyr Leu Asp Pro Glu Ala Ala Ala Ala Gly Ala AlaAla 225 230 235 240 Val Ala Asn Gln Gly Lys Lys 245 18 744 DNASaccharomyces cerevisiae 18 atgccaaagt tagttttagt tagacacggt caatccgaatggaacgaaaa gaacttattc 60 accggttggg ttgatgttaa attgtctgcc aagggtcaacaagaagccgc tagagccggt 120 gaattgttga aggaaaagaa ggtctaccca gacgtcttgtacacttccaa gttgtccaga 180 gctatccaaa ctgctaacat tgctttggaa aaggctgacagattatggat tccagtcaac 240 agatcctgga gattgaacga aagacattac ggtgacttacaaggtaagga caaggctgaa 300 actttgaaga agttcggtga agaaaaattc aacacctacagaagatcctt cgatgttcca 360 cctcccccaa tcgacgcttc ttctccattc tctcaaaagggtgatgaaag atacaagtac 420 gttgacccaa atgtcttgcc agaaactgaa tctttggctttggtcattga cagattgttg 480 ccatactggc aagatgtcat tgccaaggac ttgttgagtggtaagaccgt catgatcgcc 540 gctcacggta actccttgag aggtttggtt aagcacttggaaggtatctc tgatgctgac 600 attgctaagt tgaacatccc aactggtatt ccattggtcttcgaattgga cgaaaacttg 660 aagccatcta agccatctta ctacttggac ccagaagctgccgctgctgg tgccgctgct 720 gttgccaacc aaggtaagaa ataa 744 19 327 PRTSaccharomyces cerevisiae 19 Met Ser Ser Ser Glu Asp Glu Asp Asp Lys PheLeu Tyr Gly Ser Asp 1 5 10 15 Ser Glu Leu Ala Leu Pro Ser Ser Lys ArgSer Arg Asp Asp Glu Ala 20 25 30 Asp Ala Gly Ala Ser Ser Asn Pro Asp IleVal Lys Arg Gln Lys Phe 35 40 45 Asp Ser Pro Val Glu Glu Thr Pro Ala ThrAla Arg Asp Asp Arg Ser 50 55 60 Asp Glu Asp Ile Tyr Ser Asp Ser Ser AspAsp Asp Ser Asp Ser Asp 65 70 75 80 Leu Glu Val Ile Ile Ser Leu Gly ProAsp Pro Thr Arg Leu Asp Ala 85 90 95 Lys Leu Leu Asp Ser Tyr Ser Thr AlaAla Thr Ser Ser Ser Lys Asp 100 105 110 Val Ile Ser Val Ala Thr Asp ValSer Asn Thr Ile Thr Lys Thr Ser 115 120 125 Asp Glu Arg Leu Ile Thr GluGly Glu Ala Asn Gln Gly Val Thr Ala 130 135 140 Thr Thr Val Lys Ala ThrGlu Ser Asp Gly Asn Val Pro Lys Ala Met 145 150 155 160 Thr Gly Ser IleAsp Leu Asp Lys Glu Gly Ile Phe Asp Ser Val Gly 165 170 175 Ile Thr ThrIle Asp Pro Glu Val Leu Lys Glu Lys Pro Trp Arg Gln 180 185 190 Pro GlyAla Asn Leu Ser Asp Tyr Phe Asn Tyr Gly Phe Asn Glu Phe 195 200 205 ThrTrp Met Glu Tyr Leu His Arg Gln Glu Lys Leu Gln Gln Asp Tyr 210 215 220Asn Pro Arg Arg Ile Leu Met Gly Leu Leu Ser Leu Gln Gln Gln Gly 225 230235 240 Lys Leu Asn Ser Ala Asn Asp Thr Asp Ser Asn Leu Gly Asn Ile Ile245 250 255 Asp Asn Asn Asn Asn Val Asn Asn Ala Asn Met Ser Asn Leu AsnSer 260 265 270 Asn Met Gly Asn Ser Met Ser Gly Thr Pro Asn Pro Pro AlaPro Pro 275 280 285 Met His Pro Ser Phe Pro Pro Leu Pro Met Phe Gly SerPhe Pro Pro 290 295 300 Phe Pro Met Pro Gly Met Met Pro Pro Met Asn GlnGln Pro Asn Gln 305 310 315 320 Asn Gln Asn Gln Asn Ser Lys 325 20 984DNA Saccharomyces cerevisiae 20 atgagctcca gtgaagacga agacgacaagttcttgtatg gttccgactc cgaattagca 60 ctaccttcat ctaaacgatc aagagatgatgaagcagacg caggtgcgtc cagtaatcct 120 gatatagtta aaaggcaaaa attcgactctcccgtggaag aaactccagc tactgccaga 180 gatgatcgtt ctgatgaaga tatctactctgactcctcag atgacgatag tgattctgac 240 ctagaggtta tcataagtct gggtcctgaccctactaggt tagatgcaaa actactcgat 300 tcttattcta ccgcagcgac atcttcaagcaaagacgtaa ttagcgtagc tacagatgta 360 tccaatacca tcacaaagac atcagatgaaagactaataa cagaaggaga agcaaatcaa 420 ggtgtaacgg caacgaccgt aaaagctacagagagcgatg gaaatgtacc gaaagcaatg 480 actggttcta tagacctgga taaagagggaatctttgata gtgttggcat aacgacaata 540 gatcctgaag tattaaagga gaaaccctggaggcaaccgg gggccaactt aagtgattat 600 ttcaattacg gttttaacga atttacctggatggagtatt tacatagaca ggaaaaacta 660 caacaagatt ataatcctag gaggatcctaatgggcctat tatccctcca acagcaaggg 720 aagttgaatt ccgcgaatga tacagactcaaacctcggta atataattga taacaacaac 780 aacgtaaaca atgcaaatat gtctaatctgaacagtaata tgggtaatag tatgtctgga 840 acaccaaacc ctcccgctcc accaatgcatccaagcttcc cacccttacc tatgtttggt 900 agctttccac cattccccat gccaggtatgatgccaccca tgaaccaaca gcctaatcaa 960 aatcaaaatc aaaattcgaa atga 984 21103 PRT Saccharomyces cerevisiae 21 Met Ser Gly Arg Gly Lys Gly Gly LysGly Leu Gly Lys Gly Gly Ala 1 5 10 15 Lys Arg His Arg Lys Ile Leu ArgAsp Asn Ile Gln Gly Ile Thr Lys 20 25 30 Pro Ala Ile Arg Arg Leu Ala ArgArg Gly Gly Val Lys Arg Ile Ser 35 40 45 Gly Leu Ile Tyr Glu Glu Val ArgAla Val Leu Lys Ser Phe Leu Glu 50 55 60 Ser Val Ile Arg Asp Ser Val ThrTyr Thr Glu His Ala Lys Arg Lys 65 70 75 80 Thr Val Thr Ser Leu Asp ValVal Tyr Ala Leu Lys Arg Gln Gly Arg 85 90 95 Thr Leu Tyr Gly Phe Gly Gly100 22 312 DNA Saccharomyces cerevisiae 22 atgtccggta gaggtaaaggtggtaaaggt ctaggaaaag gtggtgccaa gcgtcacaga 60 aagattctaa gagataacattcaaggtatc actaagccag ctatcagaag attagctaga 120 agaggtggtg tcaagcgtatttctggtttg atctacgaag aagtcagagc cgtcttgaaa 180 tccttcttgg aatccgtcatcagggactct gttacttaca ctgaacacgc caagagaaag 240 actgttactt ctttggatgttgtttatgct ttgaagagac aaggtagaac cttatatggt 300 ttcggtggtt aa 312 23 132PRT Saccharomyces cerevisiae 23 Met Ser Gly Gly Lys Gly Gly Lys Ala GlySer Ala Ala Lys Ala Ser 1 5 10 15 Gln Ser Arg Ser Ala Lys Ala Gly LeuThr Phe Pro Val Gly Arg Val 20 25 30 His Arg Leu Leu Arg Arg Gly Asn TyrAla Gln Arg Ile Gly Ser Gly 35 40 45 Ala Pro Val Tyr Leu Thr Ala Val LeuGlu Tyr Leu Ala Ala Glu Ile 50 55 60 Leu Glu Leu Ala Gly Asn Ala Ala ArgAsp Asn Lys Lys Thr Arg Ile 65 70 75 80 Ile Pro Arg His Leu Gln Leu AlaIle Arg Asn Asp Asp Glu Leu Asn 85 90 95 Lys Leu Leu Gly Asn Val Thr IleAla Gln Gly Gly Val Leu Pro Asn 100 105 110 Ile His Gln Asn Leu Leu ProLys Lys Ser Ala Lys Ala Thr Lys Ala 115 120 125 Ser Gln Glu Leu 130 24399 DNA Saccharomyces cerevisiae 24 atgtccggtg gtaaaggtgg taaagctggttcagctgcta aagcttctca atctagatct 60 gctaaggctg gtttgacatt cccagtcggtagagtgcaca gattgctaag aagaggtaac 120 tacgcccaaa gaattggttc tggtgctccagtctacttga ctgctgtctt ggaatatttg 180 gccgctgaaa ttttagaatt agctggtaatgctgctaggg ataacaagaa gaccagaatt 240 attccaagac atttgcaatt ggctatcagaaatgatgacg aattgaacaa gctattgggt 300 aacgttacca ttgcccaagg tggtgttttgccaaacatcc atcaaaactt gttgccaaag 360 aagtctgcca aggctaccaa ggcttctcaagaattataa 399 25 705 PRT Saccharomyces cerevisiae 25 Met Ala Gly Glu ThrPhe Glu Phe Gln Ala Glu Ile Thr Gln Leu Met 1 5 10 15 Ser Leu Ile IleAsn Thr Val Tyr Ser Asn Lys Glu Ile Phe Leu Arg 20 25 30 Glu Leu Ile SerAsn Ala Ser Asp Ala Leu Asp Lys Ile Arg Tyr Gln 35 40 45 Ala Leu Ser AspPro Lys Gln Leu Glu Thr Glu Pro Asp Leu Phe Ile 50 55 60 Arg Ile Thr ProLys Pro Glu Glu Lys Val Leu Glu Ile Arg Asp Ser 65 70 75 80 Gly Ile GlyMet Thr Lys Ala Glu Leu Ile Asn Asn Leu Gly Thr Ile 85 90 95 Ala Lys SerGly Thr Lys Ala Phe Met Glu Ala Leu Ser Ala Gly Ala 100 105 110 Asp ValSer Met Ile Gly Gln Phe Gly Val Gly Phe Tyr Ser Leu Phe 115 120 125 LeuVal Ala Asp Arg Val Gln Val Ile Ser Lys Asn Asn Glu Asp Glu 130 135 140Gln Tyr Ile Trp Glu Ser Asn Ala Gly Gly Ser Phe Thr Val Thr Leu 145 150155 160 Asp Glu Val Asn Glu Arg Ile Gly Arg Gly Thr Val Leu Arg Leu Phe165 170 175 Leu Lys Asp Asp Gln Leu Glu Tyr Leu Glu Glu Lys Arg Ile LysGlu 180 185 190 Val Ile Lys Arg His Ser Glu Phe Val Ala Tyr Pro Ile GlnLeu Leu 195 200 205 Val Thr Lys Glu Val Glu Lys Glu Val Pro Ile Pro GluGlu Glu Lys 210 215 220 Lys Asp Glu Glu Lys Lys Asp Glu Asp Asp Lys LysPro Lys Leu Glu 225 230 235 240 Glu Val Asp Glu Glu Glu Glu Glu Lys LysPro Lys Thr Lys Lys Val 245 250 255 Lys Glu Glu Val Gln Glu Leu Glu GluLeu Asn Lys Thr Lys Pro Leu 260 265 270 Trp Thr Arg Asn Pro Ser Asp IleThr Gln Glu Glu Tyr Asn Ala Phe 275 280 285 Tyr Lys Ser Ile Ser Asn AspTrp Glu Asp Pro Leu Tyr Val Lys His 290 295 300 Phe Ser Val Glu Gly GlnLeu Glu Phe Arg Ala Ile Leu Phe Ile Pro 305 310 315 320 Lys Arg Ala ProPhe Asp Leu Phe Glu Ser Lys Lys Lys Lys Asn Asn 325 330 335 Ile Lys LeuTyr Val Arg Arg Val Phe Ile Thr Asp Glu Ala Glu Asp 340 345 350 Leu IlePro Glu Trp Leu Ser Phe Val Lys Gly Val Val Asp Ser Glu 355 360 365 AspLeu Pro Leu Asn Leu Ser Arg Glu Met Leu Gln Gln Asn Lys Ile 370 375 380Met Lys Val Ile Arg Lys Asn Ile Val Lys Lys Leu Ile Glu Ala Phe 385 390395 400 Asn Glu Ile Ala Glu Asp Ser Glu Gln Phe Asp Lys Phe Tyr Ser Ala405 410 415 Phe Ala Lys Asn Ile Lys Leu Gly Val His Glu Asp Thr Gln AsnArg 420 425 430 Ala Ala Leu Ala Lys Leu Leu Arg Tyr Asn Ser Thr Lys SerVal Asp 435 440 445 Glu Leu Thr Ser Leu Thr Asp Tyr Val Thr Arg Met ProGlu His Gln 450 455 460 Lys Asn Ile Tyr Tyr Ile Thr Gly Glu Ser Leu LysAla Val Glu Lys 465 470 475 480 Ser Pro Phe Leu Asp Ala Leu Lys Ala LysAsn Phe Glu Val Leu Phe 485 490 495 Leu Thr Asp Pro Ile Asp Glu Tyr AlaPhe Thr Gln Leu Lys Glu Phe 500 505 510 Glu Gly Lys Thr Leu Val Asp IleThr Lys Asp Phe Glu Leu Glu Glu 515 520 525 Thr Asp Glu Glu Lys Ala GluArg Glu Lys Glu Ile Lys Glu Tyr Glu 530 535 540 Pro Leu Thr Lys Ala LeuLys Asp Ile Leu Gly Asp Gln Val Glu Lys 545 550 555 560 Val Val Val SerTyr Lys Leu Leu Asp Ala Pro Ala Ala Ile Arg Thr 565 570 575 Gly Gln PheGly Trp Ser Ala Asn Met Glu Arg Ile Met Lys Ala Gln 580 585 590 Ala LeuArg Asp Ser Ser Met Ser Ser Tyr Met Ser Ser Lys Lys Thr 595 600 605 PheGlu Ile Ser Pro Lys Ser Pro Ile Ile Lys Glu Leu Lys Lys Arg 610 615 620Val Asp Glu Gly Gly Ala Gln Asp Lys Thr Val Lys Asp Leu Thr Asn 625 630635 640 Leu Leu Phe Glu Thr Ala Leu Leu Thr Ser Gly Phe Ser Leu Glu Glu645 650 655 Pro Thr Ser Phe Ala Ser Arg Ile Asn Arg Leu Ile Ser Leu GlyLeu 660 665 670 Asn Ile Asp Glu Asp Glu Glu Thr Glu Thr Ala Pro Glu AlaSer Thr 675 680 685 Glu Ala Pro Val Glu Glu Val Pro Ala Asp Thr Glu MetGlu Glu Val 690 695 700 Asp 705 26 2000 DNA Saccharomyces cerevisiae 26atggctggtg aaacttttga atttcaagct gaaatcactc agttgatgag tttgatcatc 60aacactgtct attctaacaa ggaaattttc ttgagagaac tgatctctaa cgcctccgat 120gctttagaca aaattagata ccaagctttg tctgatccaa agcaattgga aaccgaacca 180gatttgttca ttagaatcac cccaaaacca gaagaaaaag ttttggaaat cagagattct 240ggtattggta tgaccaaggc tgaattgatt aacaatttgg gtaccattgc taagtctggt 300actaaagctt tcatggaagc tctatctgct ggtgccgatg tatccatgat tggtcaattc 360ggtgttggtt tttactcttt attcttagtc gccgacagag ttcaagttat ttccaagaac 420aatgaggacg aacaatatat ttgggaatct aatgccggtg gttctttcac cgttactttg 480gacgaagtta acgaaagaat tggtagaggt accgtcttga gattattctt gaaagatgac 540caattggagt acttggaaga aaagagaatt aaagaagtca tcaagagaca ttctgaattc 600gttgcttacc ctatccaact tctagtcacc aaggaagtcg aaaaggaagt tccaattcca 660gaagaagaaa agaaagacga ggaaaagaag gatgaagatg acaagaaacc aaaattggaa 720gaagtcgatg aagaagaaga agaaaagaag ccaaaaacca aaaaagttaa agaagaggtt 780caagaattag aagagttgaa caagactaag ccattatgga ctagaaaccc atctgatatc 840actcaagagg aatacaatgc tttctataag tctatttcta acgactggga agacccattg 900tacgttaagc atttctctgt tgaaggtcaa ttggaattta gagctatctt gttcattcca 960aagagagcac cattcgactt atttgagagt aagaagaaga agaacaatat caagttgtac 1020gttcgtcgtg tcttcatcac tgatgaagct gaagacttga ttccagagtg gttatctttc 1080gtcaagggtg ttgttgactc tgaagattta ccattgaatt tgtccagaga aatgttacaa 1140caaaataaga ttatgaaggt tattagaaag aatattgtca agaaattgat tgaagccttc 1200aacgaaatcg ctgaagactc cgagcaattt gacaaatttt actctgcctt cgctaagaac 1260attaagctgg gtgtacatga ggacactcaa aacagagctg ctttagctaa gttgctacgt 1320tacaattcta ctaaatctgt cgatgaattg acttccttga ctgattacgt tactagaatg 1380ccagaacacc aaaagaacat ctattacatc accggtgaat ctctaaaggc agtcgaaaag 1440tctccattct tggacgcctt gaaggctaag aactttgaag ttttgttctt gaccgaccca 1500attgatgaat acgctttcac tcaattgaag gaattcgagg gtaaaacttt ggttgacatt 1560actaaagatt tcgaattgga agaaacagac gaagaaaaag ctgaaagaga gaaggagatc 1620aaagaatacg aaccattgac caaggccttg aaggatatct tgggtgacca agtggagaag 1680gttgttgttt cttacaaatt gctagatgct ccagctgcca tcagaactgg tcaattcggc 1740tggtctgcta acatggaaag aatcatgaag gctcaagcct tgagagactc ttccatgtcc 1800tcctacatgt cttccaagaa gactttcgaa atttctccaa aatctccaat tattaaggaa 1860ttgaaaaaga gagttgatga gggtggtgca caagataaga ccgtcaaaga tttgactaac 1920ttattattcg agaccgcttt gttgacttct ggtttcagtt tggaagaacc aacttctttt 1980gcatcaagaa taaatagatt 2000 27 523 PRT Saccharomyces cerevisiae 27 MetAla Ala Ile Arg Asp Tyr Lys Thr Ala Leu Asp Phe Thr Lys Ser 1 5 10 15Leu Pro Arg Pro Asp Gly Leu Ser Val Gln Glu Leu Met Asp Ser Lys 20 25 30Ile Arg Gly Gly Leu Thr Tyr Asn Asp Phe Leu Ile Leu Pro Gly Leu 35 40 45Val Asp Phe Ala Ser Ser Glu Val Ser Leu Gln Thr Lys Leu Thr Arg 50 55 60Asn Ile Thr Leu Asn Ile Pro Leu Val Ser Ser Pro Met Asp Thr Val 65 70 7580 Thr Glu Ser Glu Met Ala Thr Phe Met Ala Leu Leu Gly Gly Ile Gly 85 9095 Phe Ile His His Asn Cys Thr Pro Glu Asp Gln Ala Asp Met Val Arg 100105 110 Arg Val Lys Asn Tyr Glu Asn Gly Phe Ile Asn Asn Pro Ile Val Ile115 120 125 Ser Pro Thr Thr Thr Val Gly Glu Ala Lys Ser Met Lys Glu LysTyr 130 135 140 Gly Phe Ala Gly Phe Pro Val Thr Thr Asp Gly Lys Arg AsnAla Lys 145 150 155 160 Leu Val Gly Val Ile Thr Ser Arg Asp Ile Gln PheVal Glu Asp Asn 165 170 175 Ser Leu Leu Val Gln Asp Val Met Thr Lys AsnPro Val Thr Gly Ala 180 185 190 Gln Gly Ile Thr Leu Ser Glu Gly Asn GluIle Leu Lys Lys Ile Lys 195 200 205 Lys Gly Arg Leu Leu Val Val Asp GluLys Gly Asn Leu Val Ser Met 210 215 220 Leu Ser Arg Thr Asp Leu Met LysAsn Gln Asn Tyr Pro Leu Ala Ser 225 230 235 240 Lys Ser Ala Asn Thr LysGln Leu Leu Cys Gly Ala Ser Ile Gly Thr 245 250 255 Met Asp Ala Asp LysGlu Arg Leu Arg Leu Leu Val Lys Ala Gly Leu 260 265 270 Asp Val Val IleLeu Asp Ser Ser Gln Gly Asn Ser Ile Phe Glu Leu 275 280 285 Asn Met LeuLys Trp Val Lys Glu Ser Phe Pro Gly Leu Glu Val Ile 290 295 300 Ala GlyAsn Val Val Thr Arg Glu Gln Ala Ala Asn Leu Ile Ala Ala 305 310 315 320Gly Ala Asp Gly Leu Arg Ile Gly Met Gly Thr Gly Ser Ile Cys Ile 325 330335 Thr Gln Glu Val Met Ala Cys Gly Arg Pro Gln Gly Thr Ala Val Tyr 340345 350 Asn Val Cys Glu Phe Ala Asn Gln Phe Gly Val Pro Cys Met Ala Asp355 360 365 Gly Gly Val Gln Asn Ile Gly His Ile Thr Lys Ala Leu Ala LeuGly 370 375 380 Ser Ser Thr Val Met Met Gly Gly Met Leu Ala Gly Thr ThrGlu Ser 385 390 395 400 Pro Gly Glu Tyr Phe Tyr Gln Asp Gly Lys Arg LeuLys Ala Tyr Arg 405 410 415 Gly Met Gly Ser Ile Asp Ala Met Gln Lys ThrGly Thr Lys Gly Asn 420 425 430 Ala Ser Thr Ser Arg Tyr Phe Ser Glu SerAsp Ser Val Leu Val Ala 435 440 445 Gln Gly Val Ser Gly Ala Val Val AspLys Gly Ser Ile Lys Lys Phe 450 455 460 Ile Pro Tyr Leu Tyr Asn Gly LeuGln His Ser Cys Gln Asp Ile Gly 465 470 475 480 Cys Arg Ser Leu Thr LeuLeu Lys Asn Asn Val Gln Arg Gly Lys Val 485 490 495 Arg Phe Glu Phe ArgThr Ala Ser Ala Gln Leu Glu Gly Gly Val His 500 505 510 Asn Leu His SerTyr Glu Lys Arg Leu His Asn 515 520 28 1572 DNA Saccharomyces cerevisiae28 atggccgcca ttagagacta caagaccgca ctagacttta ccaagagcct accaagaccg 60gatggtttgt cagtgcagga attgatggac tccaagatca gaggtgggtt gacttataac 120gattttttaa tcttaccagg tttagtcgat tttgcgtcct ctgaagttag cctacagacc 180aagctaacca ggaatattac tttaaatatt ccattagttt cctctccaat ggacacggtg 240acagagtcag aaatggccac ttttatggct ctgttgggtg gtatcggttt cattcaccat 300aactgtaccc cagaggacca agctgacatg gtcagaagag tcaagaacta tgaaaatggg 360tttattaaca accctatagt gatttctcca actacgaccg ttggtgaagc taagagcatg 420aaggaaaagt atggatttgc aggcttccct gtcacgacag atggaaagag aaatgcaaag 480ttggtgggtg tcatcacctc tcgtgatata caattcgttg aggacaactc tttactcgtt 540caggatgtca tgaccaaaaa ccctgttacc ggcgcacaag gtatcacatt atcagaaggt 600aacgaaattc taaagaaaat caaaaagggt aggctattgg ttgttgatga aaagggtaac 660ttagtttcta tgctttcccg aactgattta atgaaaaatc agaactaccc attagcgtcc 720aaatctgcca acaccaagca actgttatgt ggtgcttcta ttgggactat ggacgctgat 780aaagaaagac taagattatt ggtaaaagct gggttggatg tcgtcatatt ggattcatcc 840caaggaaact ctatcttcga attgaacatg ctcaagtggg tcaaagagag tttcccaggt 900ctggaagtca tcgctggtaa cgttgtcacc agggaacaag ctgccaattt gattgctgcc 960ggtgcggacg gtttgagaat tggtatggga actggctcta tttgtattac ccaagaagtt 1020atggcttgtg gtaggccaca aggtacagcc gtctacaatg tgtgtgaatt tgctaaccaa 1080ttcggtgttc catgtatggc tgatggtggt gttcaaaaca ttggtcatat taccaaagct 1140ttggctcttg gttcttctac tgttatgatg ggtggtatgt tggccggtac taccgaatca 1200ccaggtgaat atttctatca agatggtaaa agattgaagg cgtatcgtgg tatgggctcc 1260attgacgcca tgcaaaagac tggtaccaaa ggtaatgcat ctacctcccg ttacttttcc 1320gaatcagaca gtgttttggt cgcacaaggt gtctccggtg ctgtcgttga caaaggatcc 1380attaagaaat ttattccata tttgtacaat ggtttacaac attcctgtca agacatcggc 1440tgtaggtcgc taactttatt aaagaataat gttcaaaggg gtaaagttag atttgaattc 1500agaaccgctt ctgctcaact agaaggtggc gttcataatt tacattctta cgaaaagcgt 1560ttacataact ga 1572 29 524 PRT Saccharomyces cerevisiae 29 Met Ser AlaAla Pro Leu Asp Tyr Lys Lys Ala Leu Glu His Leu Lys 1 5 10 15 Thr TyrSer Ser Lys Asp Gly Leu Ser Val Gln Glu Leu Met Asp Ser 20 25 30 Thr ThrArg Gly Gly Leu Thr Tyr Asn Asp Phe Leu Val Leu Pro Gly 35 40 45 Leu ValAsn Phe Pro Ser Ser Ala Val Ser Leu Gln Thr Lys Leu Thr 50 55 60 Lys LysIle Thr Leu Asn Thr Pro Phe Val Ser Ser Pro Met Asp Thr 65 70 75 80 ValThr Glu Ala Asp Met Ala Ile Tyr Met Ala Leu Leu Gly Gly Ile 85 90 95 GlyPhe Ile His His Asn Cys Thr Pro Lys Glu Gln Ala Ser Met Val 100 105 110Lys Lys Val Lys Met Phe Glu Asn Gly Phe Ile Asn Ser Pro Ile Val 115 120125 Ile Ser Pro Thr Thr Thr Val Gly Glu Val Lys Val Met Lys Arg Lys 130135 140 Phe Gly Phe Ser Gly Phe Pro Val Thr Glu Asp Gly Lys Cys Pro Gly145 150 155 160 Lys Leu Val Gly Leu Val Thr Ser Arg Asp Ile Gln Phe LeuGlu Asp 165 170 175 Asp Ser Leu Val Val Ser Glu Val Met Thr Lys Asn ProVal Thr Gly 180 185 190 Ile Lys Gly Ile Thr Leu Lys Glu Gly Asn Glu IleLeu Lys Gln Thr 195 200 205 Lys Lys Gly Lys Leu Leu Ile Val Asp Asp AsnGly Asn Leu Val Ser 210 215 220 Met Leu Ser Arg Ala Asp Leu Met Lys AsnGln Asn Tyr Pro Leu Ala 225 230 235 240 Ser Lys Ser Ala Thr Thr Lys GlnLeu Leu Cys Gly Ala Ala Ile Gly 245 250 255 Thr Ile Glu Ala Asp Lys GluArg Leu Arg Leu Leu Val Glu Ala Gly 260 265 270 Leu Asp Val Val Ile LeuAsp Ser Ser Gln Gly Asn Ser Val Phe Gln 275 280 285 Leu Asn Met Ile LysTrp Ile Lys Glu Thr Phe Pro Asp Leu Glu Ile 290 295 300 Ile Ala Gly AsnVal Ala Thr Arg Glu Gln Ala Ala Asn Leu Ile Ala 305 310 315 320 Ala GlyAla Asp Gly Leu Arg Ile Gly Met Gly Ser Gly Ser Ile Cys 325 330 335 IleThr Gln Glu Val Met Ala Cys Gly Arg Pro Gln Gly Thr Ala Val 340 345 350Tyr Asn Val Cys Gln Phe Ala Asn Gln Phe Gly Val Pro Cys Met Ala 355 360365 Asp Gly Gly Val Gln Asn Ile Gly His Ile Thr Lys Ala Leu Ala Leu 370375 380 Gly Ser Ser Thr Val Met Met Gly Gly Met Leu Ala Gly Thr Thr Glu385 390 395 400 Ser Pro Gly Glu Tyr Phe Tyr Lys Asp Gly Lys Arg Leu LysAla Tyr 405 410 415 Arg Gly Met Gly Ser Ile Asp Ala Met Gln Lys Thr GlyAsn Lys Gly 420 425 430 Asn Ala Ser Thr Ser Arg Tyr Phe Ser Glu Ser AspSer Val Leu Val 435 440 445 Ala Gln Gly Val Ser Gly Ala Val Val Asp LysGly Ser Ile Lys Lys 450 455 460 Phe Ile Pro Tyr Leu Tyr Asn Gly Leu GlnHis Ser Cys Gln Asp Ile 465 470 475 480 Gly Cys Glu Ser Leu Thr Ser LeuLys Glu Asn Val Gln Asn Gly Glu 485 490 495 Val Arg Phe Glu Phe Arg ThrAla Ser Ala Gln Leu Glu Gly Gly Val 500 505 510 His Asn Leu His Ser TyrGlu Lys Arg Leu Tyr Asn 515 520 30 1575 DNA Saccharomyces cerevisiae 30atgagtgctg ctccattgga ttacaaaaag gctttagaac atttgaagac atacagttcc 60aaggacggtc tttctgtcca ggaattgatg gactccacaa ctagaggtgg gttgacctac 120aatgattttt tggtcttacc aggcttggtc aatttcccat cttctgctgt cagtttgcaa 180accaaattga ccaagaaaat cactttgaac actccttttg tctcttctcc tatggacaca 240gtcactgaag ctgatatggc tatttatatg gctttattgg gtggtattgg tttcatccat 300cacaactgta ctccaaagga acaggcttcc atggtcaaga aagttaaaat gtttgaaaac 360ggtttcatca attctccaat agtaatttct ccaaccacca ctgttggtga agttaaggtt 420atgaagagaa agtttggttt ctctggcttc ccagttactg aagacggtaa gtgtccaggt 480aagttagttg ggttagtcac ctctcgtgat atacaattct tagaagatga ttctttggtt 540gtttctgaag tcatgactaa aaatccagtt actggcatca agggtattac tttgaaagaa 600ggtaatgaaa tcctaaaaca aaccaagaaa ggtaaattgc ttatcgttga tgataacggt 660aacctcgttt ccatgttgtc aagagcggat ttgatgaaga atcaaaacta cccgttagct 720tctaaatccg ccaccaccaa gcaattgcta tgtggtgctg caattggtac tatcgaagct 780gataaggaaa gattaagact attagtcgaa gcaggtttgg atgttgttat cttagattcc 840tctcaaggta actctgtttt ccaattgaac atgatcaaat ggattaaaga aactttccca 900gatttggaaa tcattgctgg taacgttgcc accagagaac aagctgctaa cttgattgct 960gccggtgccg atggtttaag aattggtatg ggttctgggt ctatttgtat cactcaagaa 1020gttatggctt gtggtagacc acaaggtaca gctgtctaca acgtttgtca atttgctaac 1080caatttggcg ttccatgtat ggctgatggt ggtgtccaaa acattggcca catcaccaag 1140gctttggccc ttggttcctc caccgtcatg atgggtggta tgttagctgg tactaccgag 1200tcaccaggtg aatacttcta caaggatggt aagagattga aggcttatcg tggtatgggg 1260tccattgacg ccatgcaaaa gaccggtaac aagggtaatg cctctacttc tcgttatttc 1320tctgagtcag acagtgtctt ggttgcacaa ggtgtttctg gggctgtcgt agacaaaggt 1380tccatcaaga agtttattcc atatttgtac aacggtttac aacattcttg tcaagacatt 1440ggttgcgaat ctctaacttc attgaaagag aatgttcaaa atggtgaagt tagatttgaa 1500ttcagaaccg cttctgctca attagaaggt ggtgtccata atttgcactc ctatgaaaaa 1560cgtctataca attga 1575 31 767 PRT Saccharomyces cerevisiae 31 Met Val GlnSer Ala Val Leu Gly Phe Pro Arg Ile Gly Pro Asn Arg 1 5 10 15 Glu LeuLys Lys Ala Thr Glu Gly Tyr Trp Asn Gly Lys Ile Thr Val 20 25 30 Asp GluLeu Phe Lys Val Gly Lys Asp Leu Arg Thr Gln Asn Trp Lys 35 40 45 Leu GlnLys Glu Ala Gly Val Asp Ile Ile Pro Ser Asn Asp Phe Ser 50 55 60 Phe TyrAsp Gln Val Leu Asp Leu Ser Leu Leu Phe Asn Val Ile Pro 65 70 75 80 AspArg Tyr Thr Lys Tyr Asp Leu Ser Pro Ile Asp Thr Leu Phe Ala 85 90 95 MetGly Arg Gly Leu Gln Arg Lys Ala Thr Glu Thr Glu Lys Ala Val 100 105 110Asp Val Thr Ala Leu Glu Met Val Lys Trp Phe Asp Ser Asn Tyr His 115 120125 Tyr Val Arg Pro Thr Phe Ser Lys Thr Thr Gln Phe Lys Leu Asn Gly 130135 140 Gln Lys Pro Val Asp Glu Phe Leu Glu Ala Lys Glu Leu Gly Ile His145 150 155 160 Thr Arg Pro Val Leu Leu Gly Pro Val Ser Tyr Leu Phe LeuGly Lys 165 170 175 Ala Asp Lys Asp Ser Leu Asp Leu Glu Pro Leu Ser LeuLeu Glu Gln 180 185 190 Leu Leu Pro Leu Tyr Thr Glu Ile Leu Ser Lys LeuAla Ser Ala Gly 195 200 205 Ala Thr Glu Val Gln Ile Asp Glu Pro Val LeuVal Leu Asp Leu Pro 210 215 220 Ala Asn Ala Gln Ala Ala Ile Lys Lys AlaTyr Thr Tyr Phe Gly Glu 225 230 235 240 Gln Ser Asn Leu Pro Lys Ile ThrLeu Ala Thr Tyr Phe Gly Thr Val 245 250 255 Val Pro Asn Leu Asp Ala IleLys Gly Leu Pro Val Ala Ala Leu His 260 265 270 Val Asp Phe Val Arg AlaPro Glu Gln Phe Asp Glu Val Val Ala Ala 275 280 285 Ile Gly Asn Lys GlnThr Leu Ser Val Gly Ile Val Asp Gly Arg Asn 290 295 300 Ile Trp Lys AsnAsp Phe Lys Lys Ser Ser Ala Ile Val Asn Lys Ala 305 310 315 320 Ile GluLys Leu Gly Ala Asp Arg Val Val Val Ala Thr Ser Ser Ser 325 330 335 LeuLeu His Thr Pro Val Asp Leu Asn Asn Glu Thr Lys Leu Asp Ala 340 345 350Glu Ile Lys Gly Phe Phe Ser Phe Ala Thr Gln Lys Leu Asp Glu Val 355 360365 Val Val Ile Thr Lys Asn Val Ser Gly Gln Asp Val Ala Ala Ala Leu 370375 380 Glu Ala Asn Ala Lys Ser Val Glu Ser Arg Gly Lys Ser Lys Phe Ile385 390 395 400 His Asp Ala Ala Val Lys Ala Arg Val Ala Ser Ile Asp GluLys Met 405 410 415 Ser Thr Arg Ala Ala Pro Phe Glu Gln Arg Leu Pro GluGln Gln Lys 420 425 430 Val Phe Asn Leu Pro Leu Phe Pro Thr Thr Thr IleGly Ser Phe Pro 435 440 445 Gln Thr Lys Asp Ile Arg Ile Asn Arg Asn LysPhe Asn Lys Gly Thr 450 455 460 Ile Ser Ala Glu Glu Tyr Glu Lys Phe IleAsn Ser Glu Ile Glu Lys 465 470 475 480 Val Ile Arg Phe Gln Glu Glu IleGly Leu Asp Val Leu Val His Gly 485 490 495 Glu Pro Glu Arg Asn Asp MetVal Gln Tyr Phe Gly Glu Gln Ile Asn 500 505 510 Gly Tyr Ala Phe Thr ValAsn Gly Trp Val Gln Ser Tyr Gly Ser Arg 515 520 525 Tyr Val Arg Pro ProIle Ile Val Gly Asp Leu Ser Arg Pro Lys Ala 530 535 540 Met Ser Val LysGlu Ser Val Tyr Ala Gln Ser Ile Thr Ser Lys Pro 545 550 555 560 Val LysGly Met Leu Thr Gly Pro Ile Thr Cys Leu Arg Trp Ser Phe 565 570 575 ProArg Asp Asp Val Asp Gln Lys Thr Gln Ala Met Gln Leu Ala Leu 580 585 590Ala Leu Arg Asp Glu Val Asn Asp Leu Glu Ala Ala Gly Ile Lys Val 595 600605 Ile Gln Val Asp Glu Pro Ala Leu Arg Glu Gly Leu Pro Leu Arg Glu 610615 620 Gly Thr Glu Arg Ser Ala Tyr Tyr Thr Trp Ala Ala Glu Ala Phe Arg625 630 635 640 Val Ala Thr Ser Gly Val Ala Asn Lys Thr Gln Ile His SerHis Phe 645 650 655 Cys Tyr Ser Asp Leu Asp Pro Asn His Ile Lys Ala LeuAsp Ala Asp 660 665 670 Val Val Ser Ile Glu Phe Ser Lys Lys Asp Asp AlaAsn Tyr Ile Ala 675 680 685 Glu Phe Lys Asn Tyr Pro Asn His Ile Gly LeuGly Leu Phe Asp Ile 690 695 700 His Ser Pro Arg Ile Pro Ser Lys Asp GluPhe Ile Ala Lys Ile Ser 705 710 715 720 Thr Ile Leu Lys Ser Tyr Pro AlaGlu Lys Phe Trp Val Asn Pro Asp 725 730 735 Cys Gly Leu Lys Thr Arg GlyTrp Glu Glu Thr Arg Leu Ser Leu Thr 740 745 750 His Met Val Glu Ala AlaLys Tyr Phe Arg Glu Gln Tyr Lys Asn 755 760 765 32 2304 DNASaccharomyces cerevisiae 32 atggttcaat ctgctgtctt agggttccca agaatcggtccaaacagaga attaaagaag 60 gccactgaag gttactggaa cggtaaaatc actgtcgatgaattattcaa ggtcggtaag 120 gatttgagaa ctcaaaactg gaagttgcaa aaggaggctggtgttgatat catcccatcc 180 aatgacttct ccttttacga ccaagttttg gatttgtctttgttgttcaa tgtcattcca 240 gaccgttaca ctaagtacga tctatctcca atcgacactttgtttgctat gggtagaggt 300 ttacaaagaa aggccactga aactgaaaag gctgtcgacgtcactgcttt ggaaatggtt 360 aaatggttcg actctaacta ccattacgtt agaccaactttctccaagac cactcaattt 420 aagttgaacg gccaaaagcc agttgacgaa tttttggaagccaaggagtt aggtattcat 480 actagacctg tcttgttggg tccagtttct tacttattcttgggtaaggc tgacaaggat 540 tctctagatt tggaaccatt gtccctattg gaacaattgttgcctctata cactgaaatc 600 ctatctaaat tggcttctgc tggtgccact gaagttcaaattgacgaacc tgtcttagtt 660 ttggacttgc ctgccaacgc ccaagccgcc attaagaaggcttacactta cttcggtgaa 720 caaagcaatc taccaaagat tactttggct acttacttcggtaccgttgt ccctaactta 780 gacgccatca agggcttgcc agttgctgcc ttacacgttgactttgttag agctccagaa 840 caatttgatg aagtcgttgc cgccattggt aacaaacaaaccttgtccgt tggtattgtt 900 gatggtagaa acatttggaa gaatgatttc aagaagtcttccgctatcgt taacaaggct 960 attgaaaagt tgggtgctga cagagtcgtt gttgccacttcttcttctct attgcacaca 1020 ccagttgatt tgaacaacga aaccaagttg gacgctgaaatcaagggctt tttctctttc 1080 gccactcaaa aattggatga agttgttgtg atcaccaagaacgtttccgg tcaagacgtt 1140 gctgctgccc tagaagctaa cgctaaatct gttgaatccagaggtaaatc caagtttatc 1200 cacgatgctg ccgttaaggc cagagttgcc tctatcgacgaaaaaatgtc tactagagca 1260 gctccatttg aacaaagatt gcctgaacaa caaaaagtcttcaacttgcc attgttccca 1320 acaacaacta ttggttcctt ccctcaaacc aaggacatcagaattaacag aaacaaattc 1380 aacaagggca ccatctctgc tgaagaatat gaaaaattcatcaattctga aattgaaaag 1440 gtcatcagat tccaagaaga aattggtttg gatgtcttagtccacggtga accagaaaga 1500 aacgatatgg ttcaatactt cggtgaacaa atcaacggttatgctttcac tgttaacggt 1560 tgggttcaat cttacggttc cagatatgtc agaccaccaattattgttgg tgacttgtcc 1620 agaccaaagg ctatgtccgt caaggaatct gtttacgctcaatccatcac ttctaagcca 1680 gtaaagggta tgttgactgg tccaattacc tgtttgagatggtctttccc aagagacgat 1740 gtcgaccaaa aaactcaagc tatgcaatta gctttggctttgagagatga agtcaatgat 1800 ttggaagctg ccggtatcaa ggttatccaa gttgatgaaccagctttaag agaaggttta 1860 ccattgagag aaggtactga gagatctgct tactacacctgggctgccga agctttcaga 1920 gttgctactt ctggtgttgc taacaagact caaatacactctcatttctg ttactctgac 1980 ttggatccaa accatatcaa ggctttggat gctgatgttgtttccatcga attctctaag 2040 aaggacgatg ctaactacat tgctgaattc aaaaactatccaaaccacat tggtctaggt 2100 ttattcgata ttcattctcc aagaattcca tcaaaggatgaatttatcgc caagatttca 2160 accatcttga agagctaccc agctgaaaag ttctgggttaacccagattg tggtttgaag 2220 actagaggct gggaagaaac tagattgtct ttgactcatatggtcgaagc cgccaagtac 2280 ttccgtgaac aatacaagaa ttaa 2304 33 577 PRTSaccharomyces cerevisiae 33 Met Ala Asp Ile Thr Asp Lys Thr Ala Glu GlnLeu Glu Asn Leu Asn 1 5 10 15 Ile Gln Asp Asp Gln Lys Gln Ala Ala ThrGly Ser Glu Ser Gln Ser 20 25 30 Val Glu Asn Ser Ser Ala Ser Leu Tyr ValGly Asp Leu Glu Pro Ser 35 40 45 Val Ser Glu Ala His Leu Tyr Asp Ile PheSer Pro Ile Gly Ser Val 50 55 60 Ser Ser Ile Arg Val Cys Arg Asp Ala IleThr Lys Thr Ser Leu Gly 65 70 75 80 Tyr Ala Tyr Val Asn Phe Asn Asp HisGlu Ala Gly Arg Lys Ala Ile 85 90 95 Glu Gln Leu Asn Tyr Thr Pro Ile LysGly Arg Leu Cys Arg Ile Met 100 105 110 Trp Ser Gln Arg Asp Pro Ser LeuArg Lys Lys Gly Ser Gly Asn Ile 115 120 125 Phe Ile Lys Asn Leu His ProAsp Ile Asp Asn Lys Ala Leu Tyr Asp 130 135 140 Thr Phe Ser Val Phe GlyAsp Ile Leu Ser Ser Lys Ile Ala Thr Asp 145 150 155 160 Glu Asn Gly LysSer Lys Gly Phe Gly Phe Val His Phe Glu Glu Glu 165 170 175 Gly Ala AlaLys Glu Ala Ile Asp Ala Leu Asn Gly Met Leu Leu Asn 180 185 190 Gly GlnGlu Ile Tyr Val Ala Pro His Leu Ser Arg Lys Glu Arg Asp 195 200 205 SerGln Leu Glu Glu Thr Lys Ala His Tyr Thr Asn Leu Tyr Val Lys 210 215 220Asn Ile Asn Ser Glu Thr Thr Asp Glu Gln Phe Gln Glu Leu Phe Ala 225 230235 240 Lys Phe Gly Pro Ile Val Ser Ala Ser Leu Glu Lys Asp Ala Asp Gly245 250 255 Lys Leu Lys Gly Phe Gly Phe Val Asn Tyr Glu Lys His Glu AspAla 260 265 270 Val Lys Ala Val Glu Ala Leu Asn Asp Ser Glu Leu Asn GlyGlu Lys 275 280 285 Leu Tyr Val Gly Arg Ala Gln Lys Lys Asn Glu Arg MetHis Val Leu 290 295 300 Lys Lys Gln Tyr Glu Ala Tyr Arg Leu Glu Lys MetAla Lys Tyr Gln 305 310 315 320 Gly Val Asn Leu Phe Val Lys Asn Leu AspAsp Ser Val Asp Asp Glu 325 330 335 Lys Leu Glu Glu Glu Phe Ala Pro TyrGly Thr Ile Thr Ser Ala Lys 340 345 350 Val Met Arg Thr Glu Asn Gly LysSer Lys Gly Phe Gly Phe Val Cys 355 360 365 Phe Ser Thr Pro Glu Glu AlaThr Lys Ala Ile Thr Glu Lys Asn Gln 370 375 380 Gln Ile Val Ala Gly LysPro Leu Tyr Val Ala Ile Ala Gln Arg Lys 385 390 395 400 Asp Val Arg ArgSer Gln Leu Ala Gln Gln Ile Gln Ala Arg Asn Gln 405 410 415 Met Arg TyrGln Gln Ala Thr Ala Ala Ala Ala Ala Ala Ala Ala Gly 420 425 430 Met ProGly Gln Phe Met Pro Pro Met Phe Tyr Gly Val Met Pro Pro 435 440 445 ArgGly Val Pro Phe Asn Gly Pro Asn Pro Gln Gln Met Asn Pro Met 450 455 460Gly Gly Met Pro Lys Asn Gly Met Pro Pro Gln Phe Arg Asn Gly Pro 465 470475 480 Val Tyr Gly Val Pro Pro Gln Gly Gly Phe Pro Arg Asn Ala Asn Asp485 490 495 Asn Asn Gln Phe Tyr Gln Gln Lys Gln Arg Gln Ala Leu Gly GluGln 500 505 510 Leu Tyr Lys Lys Val Ser Ala Lys Thr Ser Asn Glu Glu AlaAla Gly 515 520 525 Lys Ile Thr Gly Met Ile Leu Asp Leu Pro Pro Gln GluVal Phe Pro 530 535 540 Leu Leu Glu Ser Asp Glu Leu Phe Glu Gln His TyrLys Glu Ala Ser 545 550 555 560 Ala Ala Tyr Glu Ser Phe Lys Lys Glu GlnGlu Gln Gln Thr Glu Gln 565 570 575 Ala 34 1734 DNA Saccharomycescerevisiae 34 atggctgata ttactgataa gacagctgaa caattggaaa acttgaatattcaagatgac 60 caaaagcaag ccgccactgg ttcagaaagc caatctgttg aaaactcttctgcatcatta 120 tatgttggtg acttagaacc ttctgtttcc gaagcccact tatatgatatcttctctcca 180 atcggttcag tctcctccat tcgtgtctgt cgtgatgcca tcactaagacctctttgggc 240 tatgcttatg ttaactttaa cgaccatgaa gccggcagaa aagcaattgagcaattgaac 300 tacactccaa tcaagggtag attatgccgt attatgtggt ctcaacgtgacccatcattg 360 agaaagaagg gttctggtaa catctttatc aagaacttgc accctgatattgacaacaag 420 gctttgtatg acactttctc tgtgtttggt gacatcttgt ccagcaagattgccaccgac 480 gaaaacggaa aatccaaggg ttttgggttt gttcacttcg aagaagaaggtgctgccaag 540 gaagctattg atgctttgaa tggtatgctg ttgaacggtc aagaaatttatgttgctcct 600 cacttgtcca gaaaggaacg tgactctcaa ttggaagaga ctaaggcacattacactaac 660 ctttatgtga aaaacatcaa ctccgaaact actgacgaac aattccaagaattgtttgcc 720 aaatttggtc caattgtttc tgcctctttg gaaaaggatg ctgatggaaaattgaagggt 780 ttcgggtttg ttaactacga aaagcatgaa gacgctgtga aagctgttgaagctttgaat 840 gactctgaac taaatggaga aaagttatac gttggtcgtg cccaaaagaagaatgaacgt 900 atgcatgtct tgaagaagca atacgaagct tacagattgg aaaaaatggccaagtaccaa 960 ggtgttaatt tgtttgttaa gaacttagat gacagcgttg atgacgaaaagttggaagaa 1020 gaatttgctc catatggtac tatcacttct gcaaaggtta tgagaaccgaaaacggtaag 1080 tctaagggtt ttggttttgt ttgtttctca actccagagg aagctactaaggccattaca 1140 gaaaagaacc aacaaattgt tgctggtaag ccattatacg ttgccattgctcaaagaaaa 1200 gacgtaagac gttctcaatt ggctcaacaa atccaagcca gaaatcaaatgagataccag 1260 caagctactg ctgccgctgc cgccgccgct gccggtatgc caggtcaattcatgcctcca 1320 atgttctatg gtgttatgcc accaagaggt gttccattca acggtccaaacccacaacaa 1380 atgaacccaa tgggcggtat gccaaagaac ggcatgccac ctcaatttagaaatggtccg 1440 gtttacggcg tccccccaca aggtggtttc ccaagaaatg ccaacgataacaaccaattt 1500 tatcaacaaa agcaaagaca agctttgggt gaacaattat acaagaaggtttctgctaag 1560 acttcaaatg aagaagcagc tggtaaaatt actggtatga ttttggatttgccacctcaa 1620 gaggtcttcc cattgttgga aagtgatgaa ttgttcgaac aacactacaaagaagcttct 1680 gctgcctatg agtctttcaa aaaggagcaa gaacaacaaa ctgagcaagcttaa 1734 35 568 PRT Saccharomyces cerevisiae 35 Met Ser Ser Gln Lys ValPhe Gly Ile Thr Gly Pro Val Ser Thr Val 1 5 10 15 Gly Ala Thr Ala AlaGlu Asn Lys Leu Asn Asp Ser Leu Ile Gln Glu 20 25 30 Leu Lys Lys Glu GlySer Phe Glu Thr Glu Gln Glu Thr Ala Asn Arg 35 40 45 Val Gln Val Leu LysIle Leu Gln Glu Leu Ala Gln Arg Phe Val Tyr 50 55 60 Glu Val Ser Lys LysLys Asn Met Ser Asp Gly Met Ala Arg Asp Ala 65 70 75 80 Gly Gly Lys IlePhe Thr Tyr Gly Ser Tyr Arg Leu Gly Val His Gly 85 90 95 Pro Gly Ser AspIle Asp Thr Leu Val Val Val Pro Lys His Val Thr 100 105 110 Arg Glu AspPhe Phe Thr Val Phe Asp Ser Leu Leu Arg Glu Arg Lys 115 120 125 Glu LeuAsp Glu Ile Ala Pro Val Pro Asp Ala Phe Val Pro Ile Ile 130 135 140 LysIle Lys Phe Ser Gly Ile Ser Ile Asp Leu Ile Cys Ala Arg Leu 145 150 155160 Asp Gln Pro Gln Val Pro Leu Ser Leu Thr Leu Ser Asp Lys Asn Leu 165170 175 Leu Arg Asn Leu Asp Glu Lys Asp Leu Arg Ala Leu Asn Gly Thr Arg180 185 190 Val Thr Asp Glu Ile Leu Glu Leu Val Pro Lys Pro Asn Val PheArg 195 200 205 Ile Ala Leu Arg Ala Ile Lys Leu Trp Ala Gln Arg Arg AlaVal Tyr 210 215 220 Ala Asn Ile Phe Gly Phe Pro Gly Gly Val Ala Trp AlaMet Leu Val 225 230 235 240 Ala Arg Ile Cys Gln Leu Tyr Pro Asn Ala CysSer Ala Val Ile Leu 245 250 255 Asn Arg Phe Phe Ile Ile Leu Ser Glu TrpAsn Trp Pro Gln Pro Val 260 265 270 Ile Leu Lys Pro Ile Glu Asp Gly ProLeu Gln Val Arg Val Trp Asn 275 280 285 Pro Lys Ile Tyr Ala Gln Asp ArgSer His Arg Met Pro Val Ile Thr 290 295 300 Pro Ala Tyr Pro Ser Met CysAla Thr His Asn Ile Thr Glu Ser Thr 305 310 315 320 Lys Lys Val Ile LeuGln Glu Phe Val Arg Gly Val Gln Ile Thr Asn 325 330 335 Asp Ile Phe SerAsn Lys Lys Ser Trp Ala Asn Leu Phe Glu Lys Asn 340 345 350 Asp Phe PhePhe Arg Tyr Lys Phe Tyr Leu Glu Ile Thr Ala Tyr Thr 355 360 365 Arg GlySer Asp Glu Gln His Leu Lys Trp Ser Gly Leu Val Glu Ser 370 375 380 LysVal Arg Leu Leu Val Met Lys Leu Glu Val Leu Ala Gly Ile Lys 385 390 395400 Ile Ala His Pro Phe Thr Lys Pro Phe Glu Ser Ser Tyr Cys Cys Pro 405410 415 Thr Glu Asp Asp Tyr Glu Met Ile Gln Asp Lys Tyr Gly Ser His Lys420 425 430 Thr Glu Thr Ala Leu Asn Ala Leu Lys Leu Val Thr Asp Glu AsnLys 435 440 445 Glu Glu Glu Ser Ile Lys Asp Ala Pro Lys Ala Tyr Leu SerThr Met 450 455 460 Tyr Ile Gly Leu Asp Phe Asn Ile Glu Asn Lys Lys GluLys Val Asp 465 470 475 480 Ile His Ile Pro Cys Thr Glu Phe Val Asn LeuCys Arg Ser Phe Asn 485 490 495 Glu Asp Tyr Gly Asp His Lys Val Phe AsnLeu Ala Leu Arg Phe Val 500 505 510 Lys Gly Tyr Asp Leu Pro Asp Glu ValPhe Asp Glu Asn Glu Lys Arg 515 520 525 Pro Ser Lys Lys Ser Lys Arg LysAsn Leu Asp Ala Arg His Glu Thr 530 535 540 Val Lys Arg Ser Lys Ser AspAla Ala Ser Gly Asp Asn Ile Asn Gly 545 550 555 560 Thr Thr Ala Ala ValAsp Val Asn 565 36 1707 DNA Saccharomyces cerevisiae 36 atgagctctcaaaaggtttt tggtattact ggacctgttt ccaccgtggg cgccacagca 60 gcagaaaataaattaaatga tagtttaatc caagaactga aaaaggaagg atcgttcgaa 120 acagagcaagaaactgccaa tagggtacaa gtgttgaaaa tattgcagga attggcacaa 180 agatttgtttatgaagtatc gaagaagaaa aatatgtcag acgggatggc aagggatgct 240 ggtgggaagatttttacgta tgggtcctat agactaggag tccatgggcc tggtagtgat 300 atcgatactttggtagttgt tccaaaacat gtaactcggg aagatttttt tacggtattt 360 gattcactactgagagagag gaaggaactg gatgaaattg cacctgtacc tgatgcgttt 420 gtcccgattatcaagataaa gttcagtggt atttctatcg atttaatctg tgcacgtcta 480 gaccaacctcaagtgccttt atccttgact ttatcagata aaaatctact gcgaaatcta 540 gacgagaaggacttgagagc tttgaatggt accagagtaa cagatgagat attagaactg 600 gtaccaaagccgaatgtttt cagaatcgct ttaagagcta ttaagctatg ggcccaaaga 660 agggctgtttatgctaatat ttttggtttt cctggtggtg tggcttgggc catgctagtg 720 gctagaatttgtcaactata ccctaacgcc tgtagcgcag ttatattgaa cagatttttc 780 atcattttgtcggaatggaa ttggccacaa cctgttatct tgaaaccaat tgaggatggc 840 ccgttacaagttcgtgtatg gaatccaaag atatatgccc aagacaggtc tcatagaatg 900 cccgtcattacaccagctta tccatcaatg tgtgctaccc ataacatcac ggaatctact 960 aaaaaagtcattttacagga attcgtaaga ggcgttcaaa ttacgaatga tattttttcc 1020 aataagaagtcctgggccaa tttattcgaa aaaaacgatt ttttctttcg atacaagttc 1080 tatttagaaattactgcata tacaaggggc agtgacgagc agcatttaaa atggagtggt 1140 cttgttgaaagtaaggtaag gcttctagtt atgaaactgg aggtgttagc tggaataaaa 1200 attgcacatcctttcaccaa accctttgaa agtagttatt gttgtccaac cgaggatgac 1260 tatgaaatgattcaagacaa atacggtagt cataaaactg agacagcact gaacgccctt 1320 aaactggtaacagatgaaaa taaagaggaa gaaagtatta aagatgcacc aaaggcatat 1380 ttaagcaccatgtacatagg ccttgacttt aatattgaaa acaaaaagga aaaagttgac 1440 attcacattccctgcactga atttgtgaat ttatgtcgaa gtttcaatga ggattatggt 1500 gaccacaaagtattcaatct agccctccgc ttcgtaaagg gttacgattt gccagatgaa 1560 gttttcgatgaaaatgaaaa gagaccatca aagaagagta aaaggaagaa tttagatgct 1620 agacatgaaaccgtgaagag atctaaatca gatgctgctt caggtgacaa catcaatggc 1680 acaaccgcagctgttgacgt aaactaa 1707 37 626 PRT Saccharomyces cerevisiae 37 Met AspHis Asp Thr Glu Val Ile Val Lys Asp Phe Asn Ser Ile Leu 1 5 10 15 GluGlu Leu Thr Phe Asn Ser Arg Pro Ile Ile Thr Thr Leu Thr Lys 20 25 30 LeuAla Glu Glu Asn Ile Ser Cys Ala Gln Tyr Phe Val Asp Ala Ile 35 40 45 GluSer Arg Ile Glu Lys Cys Met Pro Lys Gln Lys Leu Tyr Ala Phe 50 55 60 TyrAla Leu Asp Ser Ile Cys Lys Asn Val Gly Ser Pro Tyr Thr Ile 65 70 75 80Tyr Phe Ser Arg Asn Leu Phe Asn Leu Tyr Lys Arg Thr Tyr Leu Leu 85 90 95Val Asp Asn Thr Thr Arg Thr Lys Leu Ile Asn Met Phe Lys Leu Trp 100 105110 Leu Asn Pro Asn Asp Thr Gly Leu Pro Leu Phe Glu Gly Ser Ala Leu 115120 125 Glu Lys Ile Glu Gln Phe Leu Ile Lys Ala Ser Ala Leu His Gln Lys130 135 140 Asn Leu Gln Ala Met Leu Pro Thr Pro Thr Val Pro Leu Leu LeuArg 145 150 155 160 Asp Ile Asp Lys Leu Thr Cys Leu Thr Ser Glu Arg LeuLys Asn Gln 165 170 175 Pro Asn Asp Glu Lys Leu Lys Met Lys Leu Leu ValLeu Ser Gln Leu 180 185 190 Lys Gln Glu Leu Lys Arg Glu Lys Leu Thr LeuAsn Ala Leu Lys Gln 195 200 205 Val Gln Met Gln Leu Arg Gln Val Phe SerGln Asp Gln Gln Val Leu 210 215 220 Gln Glu Arg Met Arg Tyr His Glu LeuGln Gln Gln Gln Gln Gln Gln 225 230 235 240 Gln Gln Gln Gln Gln Gln GlnGln Gln Gln Gln Gln Gln Tyr His Glu 245 250 255 Thr Lys Asp Met Val GlySer Tyr Thr Gln Asn Ser Asn Ser Ala Ile 260 265 270 Pro Leu Phe Gly AsnAsn Ser Asp Thr Thr Asn Gln Gln Asn Ser Leu 275 280 285 Ser Ser Ser LeuPhe Gly Asn Ile Ser Gly Val Glu Ser Phe Gln Glu 290 295 300 Ile Glu LysLys Lys Ser Leu Asn Lys Ile Asn Asn Leu Tyr Ala Ser 305 310 315 320 LeuLys Ala Glu Gly Leu Ile Tyr Thr Pro Pro Lys Glu Ser Ile Val 325 330 335Thr Leu Tyr Lys Lys Leu Asn Gly His Ser Asn Tyr Ser Leu Asp Ser 340 345350 His Glu Lys Gln Leu Met Lys Asn Leu Pro Lys Ile Pro Leu Leu Asn 355360 365 Asp Ile Leu Ser Asp Cys Lys Ala Tyr Phe Ala Thr Val Asn Ile Asp370 375 380 Val Leu Asn Asn Pro Ser Leu Gln Leu Ser Glu Gln Thr Leu LeuGln 385 390 395 400 Glu Asn Pro Ile Val Gln Asn Asn Leu Ile His Leu LeuTyr Arg Ser 405 410 415 Lys Pro Asn Lys Cys Ser Val Cys Gly Lys Arg PheGly Asn Ser Glu 420 425 430 Ser Glu Lys Leu Leu Gln Asn Glu His Leu AspTrp His Phe Arg Ile 435 440 445 Asn Thr Arg Ile Lys Gly Ser Gln Asn ThrAla Asn Thr Gly Ile Ser 450 455 460 Asn Ser Asn Leu Asn Thr Thr Thr ThrArg Lys Asn Ile Gln Ser Arg 465 470 475 480 Asn Trp Tyr Leu Ser Asp SerGln Trp Ala Ala Phe Lys Asp Asp Glu 485 490 495 Ile Thr Ser Thr Lys HisLys Asn Asp Tyr Thr Asp Pro His Ala Asn 500 505 510 Lys Asn Ile Asp LysSer Ala Leu Asn Ile His Ala Asp Glu Asn Asp 515 520 525 Glu Gly Ser ValAsp Asn Thr Leu Gly Ser Asp Arg Ser Asn Glu Leu 530 535 540 Glu Ile ArgGly Lys Tyr Val Val Val Pro Glu Thr Ser Gln Asp Met 545 550 555 560 AlaPhe Lys Cys Pro Ile Cys Lys Glu Thr Val Thr Gly Val Tyr Asp 565 570 575Glu Glu Ser Gly Glu Trp Val Trp Lys Asn Thr Ile Glu Val Asn Gly 580 585590 Lys Tyr Phe His Ser Thr Cys Tyr His Glu Thr Ser Gln Asn Ser Ser 595600 605 Lys Ser Asn Ser Gly Lys Val Gly Leu Asp Asp Leu Lys Lys Leu Val610 615 620 Thr Lys 625 38 1881 DNA Saccharomyces cerevisiae 38atggatcacg acacagaagt tatagtcaag gatttcaaca gtattcttga agaattaact 60tttaactcga gacccattat taccactctc acgaagttag cagaagaaaa catttcgtgt 120gctcaatatt ttgttgacgc cattgagagt agaattgaaa aatgtatgcc taagcagaag 180ctatacgctt tttatgcctt ggactccatt tgtaagaatg ttggcagccc atacacaatc 240tacttcagta gaaatttgtt taacttgtac aaaagaacat acttattggt ggacaacacc 300acaagaacaa aactcatcaa tatgtttaag ctttggttga atcctaatga caccggcctg 360cctctgtttg aaggctcagc actggagaaa attgaacagt ttttaataaa ggccagtgct 420ctccaccaga aaaacctgca agcaatgtta ccaactccga ctgttcccct cctattaaga 480gacattgata aattaacttg tttaactagt gaaaggctaa agaatcaacc aaatgatgaa 540aaattaaaaa tgaaactgtt ggttttatct cagttgaaac aagaactgaa aagagaaaaa 600ctaactctca atgctctgaa acaagttcaa atgcaactaa ggcaggtctt ctcacaggac 660caacaagttt tgcaggaaag aatgaggtat cacgaacttc agcagcaaca gcaacaacag 720cagcaacagc aacaacaaca acagcagcag cagcagcagt atcacgaaac taaggatatg 780gtcggttctt acacacaaaa ctctaattca gcgattccat tgtttggaaa taattcagat 840acaacgaacc aacagaattc tttatcttcc tctctttttg gtaatatttc tggcgttgaa 900tcttttcaag aaattgagaa aaaaaagtcc ctaaacaaaa tcaataacct ctatgcatct 960cttaaggctg aagggctcat atatactcca ccaaaagaat ccatcgtaac gctttataaa 1020aagctaaatg gacactcaaa ctactctctt gattcacacg aaaaacaatt gatgaagaac 1080ctgccaaaga tacccttatt aaatgatatt ctttctgact gcaaagctta ttttgccacg 1140gttaacatcg acgttttgaa taacccatcc ctgcagctgt ccgaacaaac tcttcttcaa 1200gaaaatccca tcgtccaaaa taatcttatt cacttattat accgatccaa gccaaataaa 1260tgtagcgtgt gtggtaagag atttggcaac tcagaaagtg aaaaactact acaaaatgag 1320catttggact ggcatttcag aataaataca agaattaagg gatcacaaaa cacagcaaat 1380acaggcataa gcaactctaa cttaaataca actaccacaa ggaaaaatat tcaatcaaga 1440aactggtatt tgagtgactc tcaatgggca gccttcaaag atgatgaaat tacctccaca 1500aagcataaaa atgattatac tgatccgcat gccaataaaa atatcgataa aagtgcccta 1560aatattcacg ctgatgaaaa tgacgaaggc tctgtagaca acactttagg aagtgataga 1620agtaacgagt tggaaatacg gggaaaatat gttgttgtac cagaaacttc acaagatatg 1680gccttcaaat gtccaatttg taaggaaacc gttactggcg tttatgatga agaatctggt 1740gaatgggttt ggaaaaatac tattgaagtg aatggcaaat atttccactc gacatgttat 1800catgaaacgt cgcaaaattc aagcaaatct aatagtggca aggtcggttt ggatgactta 1860aagaaattgg tcacaaaata a 1881 39 563 PRT Saccharomyces cerevisiae 39 MetSer Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn 35 40 45Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 7580 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 9095 His Val Val Gly Val Pro Ser Ile Ser Ala Gln Ala Lys Gln Leu Leu 100105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile AlaThr 130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr ValThr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu ValAsp Leu Asn Val 165 170 175 Pro Ala Lys Leu Leu Gln Thr Pro Ile Asp MetSer Leu Lys Pro Asn 180 185 190 Asp Ala Glu Ser Glu Lys Glu Val Ile AspThr Ile Leu Ala Leu Val 195 200 205 Lys Asp Ala Lys Asn Pro Val Ile LeuAla Asp Ala Cys Cys Ser Arg 210 215 220 His Asp Val Lys Ala Glu Thr LysLys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ala Phe Val Thr ProMet Gly Lys Gly Ser Ile Asp Glu Gln His 245 250 255 Pro Arg Tyr Gly GlyVal Tyr Val Gly Thr Leu Ser Lys Pro Glu Val 260 265 270 Lys Glu Ala ValGlu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu 275 280 285 Leu Ser AspPhe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 Asn IleVal Glu Phe His Ser Asp His Met Lys Ile Arg Asn Ala Thr 305 310 315 320Phe Pro Gly Val Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Thr 325 330335 Ile Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Pro Ala Arg 340345 350 Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro Leu Lys Gln Glu355 360 365 Trp Met Trp Asn Gln Leu Gly Asn Phe Leu Gln Glu Gly Asp ValVal 370 375 380 Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln ThrThr Phe 385 390 395 400 Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val Leu TrpGly Ser Ile Gly 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala PheAla Ala Glu Glu Ile 420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe IleGly Asp Gly Ser Leu Gln 435 440 445 Leu Thr Val Gln Glu Ile Ser Thr MetIle Arg Trp Gly Leu Lys Pro 450 455 460 Tyr Leu Phe Val Leu Asn Asn AspGly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 His Gly Pro Lys Ala GlnTyr Asn Glu Ile Gln Gly Trp Asp His Leu 485 490 495 Ser Leu Leu Pro ThrPhe Gly Ala Lys Asp Tyr Glu Thr His Arg Val 500 505 510 Ala Thr Thr GlyGlu Trp Asp Lys Leu Thr Gln Asp Lys Ser Phe Asn 515 520 525 Asp Asn SerLys Ile Arg Met Ile Glu Ile Met Leu Pro Val Phe Asp 530 535 540 Ala ProGln Asn Leu Val Glu Gln Ala Lys Leu Thr Ala Ala Thr Asn 545 550 555 560Ala Lys Gln 40 1692 DNA Saccharomyces cerevisiae 40 atgtctgaaattactttggg taaatatttg ttcgaaagat taaagcaagt caacgttaac 60 accgttttcggtttgccagg tgacttcaac ttgtccttgt tggacaagat ctacgaagtt 120 gaaggtatgagatgggctgg taacgccaac gaattgaacg ctgcttacgc cgctgatggt 180 tacgctcgtatcaagggtat gtcttgtatc atcaccacct tcggtgtcgg tgaattgtct 240 gctttgaacggtattgccgg ttcttacgct gaacacgtcg gtgttttgca cgttgttggt 300 gtcccatccatctctgctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360 gacttcactgttttccacag aatgtctgcc aacatttctg aaaccactgc tatgatcact 420 gacattgctaccgccccagc tgaaattgac agatgtatca gaaccactta cgtcacccaa 480 agaccagtctacttaggttt gccagctaac ttggtcgact tgaacgtccc agctaagttg 540 ttgcaaactccaattgacat gtctttgaag ccaaacgatg ctgaatccga aaaggaagtc 600 attgacaccatcttggcttt ggtcaaggat gctaagaacc cagttatctt ggctgatgct 660 tgttgttccagacacgacgt caaggctgaa actaagaagt tgattgactt gactcaattc 720 ccagctttcgtcaccccaat gggtaagggt tccattgacg aacaacaccc aagatacggt 780 ggtgtttacgtcggtacctt gtccaagcca gaagttaagg aagccgttga atctgctgac 840 ttgattttgtctgtcggtgc tttgttgtct gatttcaaca ccggttcttt ctcttactct 900 tacaagaccaagaacattgt cgaattccac tccgaccaca tgaagatcag aaacgccact 960 ttcccaggtgtccaaatgaa attcgttttg caaaagttgt tgaccactat tgctgacgcc 1020 gctaagggttacaagccagt tgctgtccca gctagaactc cagctaacgc tgctgtccca 1080 gcttctaccccattgaagca agaatggatg tggaaccaat tgggtaactt cttgcaagaa 1140 ggtgatgttgtcattgctga aaccggtacc tccgctttcg gtatcaacca aaccactttc 1200 ccaaacaacacctacggtat ctctcaagtc ttatggggtt ccattggttt caccactggt 1260 gctaccttgggtgctgcttt cgctgctgaa gaaattgatc caaagaagag agttatctta 1320 ttcattggtgacggttcttt gcaattgact gttcaagaaa tctccaccat gatcagatgg 1380 ggcttgaagccatacttgtt cgtcttgaac aacgatggtt acaccattga aaagttgatt 1440 cacggtccaaaggctcaata caacgaaatt caaggttggg accacctatc cttgttgcca 1500 actttcggtgctaaggacta tgaaacccac agagtcgcta ccaccggtga atgggacaag 1560 ttgacccaagacaagtcttt caacgacaac tctaagatca gaatgattga aatcatgttg 1620 ccagtcttcgatgctccaca aaacttggtt gaacaagcta agttgactgc tgctaccaac 1680 gctaagcaataa 1692 41 987 PRT Saccharomyces cerevisiae 41 Met Gln Ser Gln Asp SerCys Tyr Gly Val Ala Phe Arg Ser Ile Ile 1 5 10 15 Thr Asn Asp Glu AlaLeu Phe Lys Lys Thr Ile His Phe Tyr His Thr 20 25 30 Leu Gly Phe Ala ThrVal Lys Asp Phe Asn Lys Phe Lys His Gly Glu 35 40 45 Asn Ser Leu Leu SerSer Gly Thr Ser Gln Asp Ser Leu Arg Glu Val 50 55 60 Trp Leu Glu Ser PheLys Leu Ser Glu Val Asp Ala Ser Gly Phe Arg 65 70 75 80 Ile Pro Gln GlnGlu Ala Thr Asn Lys Ala Gln Ser Gln Gly Ala Leu 85 90 95 Leu Lys Ile ArgLeu Val Met Ser Ala Pro Ile Asp Glu Thr Phe Asp 100 105 110 Thr Asn GluThr Ala Thr Ile Thr Tyr Phe Ser Thr Asp Leu Asn Lys 115 120 125 Ile ValGlu Lys Phe Pro Lys Gln Ala Glu Lys Leu Ser Asp Thr Leu 130 135 140 ValPhe Leu Lys Asp Pro Met Gly Asn Asn Ile Thr Phe Ser Gly Leu 145 150 155160 Ala Asn Ala Thr Asp Ser Ala Pro Thr Ser Lys Asp Ala Phe Leu Glu 165170 175 Ala Thr Ser Glu Asp Glu Ile Ile Ser Arg Ala Ser Ser Asp Ala Ser180 185 190 Asp Leu Leu Arg Gln Thr Leu Gly Ser Ser Gln Lys Lys Lys LysIle 195 200 205 Ala Val Met Thr Ser Gly Gly Asp Ser Pro Gly Met Asn AlaAla Val 210 215 220 Arg Ala Val Val Arg Thr Gly Ile His Phe Gly Cys AspVal Phe Ala 225 230 235 240 Val Tyr Glu Gly Tyr Glu Gly Leu Leu Arg GlyGly Lys Tyr Leu Lys 245 250 255 Lys Met Ala Trp Glu Asp Val Arg Gly TrpLeu Ser Glu Gly Gly Thr 260 265 270 Leu Ile Gly Thr Ala Arg Ser Met GluPhe Arg Lys Arg Glu Gly Arg 275 280 285 Arg Gln Ala Ala Gly Asn Leu IleSer Gln Gly Ile Asp Ala Leu Val 290 295 300 Val Cys Gly Gly Asp Gly SerLeu Thr Gly Ala Asp Leu Phe Arg His 305 310 315 320 Glu Trp Pro Ser LeuVal Asp Glu Leu Val Ala Glu Gly Arg Phe Thr 325 330 335 Lys Glu Glu ValAla Pro Tyr Lys Asn Leu Ser Ile Val Gly Leu Val 340 345 350 Gly Ser IleAsp Asn Asp Met Ser Gly Thr Asp Ser Thr Ile Gly Ala 355 360 365 Tyr SerAla Leu Glu Arg Ile Cys Glu Met Val Asp Tyr Ile Asp Ala 370 375 380 ThrAla Lys Ser His Ser Arg Ala Phe Val Val Glu Val Met Gly Arg 385 390 395400 His Cys Gly Trp Leu Ala Leu Met Ala Gly Ile Ala Thr Gly Ala Asp 405410 415 Tyr Ile Phe Ile Pro Glu Arg Ala Val Pro His Gly Lys Trp Gln Asp420 425 430 Glu Leu Lys Glu Val Cys Gln Arg His Arg Ser Lys Gly Arg ArgAsn 435 440 445 Asn Thr Ile Ile Val Ala Glu Gly Ala Leu Asp Asp Gln LeuAsn Pro 450 455 460 Val Thr Ala Asn Asp Val Lys Asp Ala Leu Ile Glu LeuGly Leu Asp 465 470 475 480 Thr Lys Val Thr Ile Leu Gly His Val Gln ArgGly Gly Thr Ala Val 485 490 495 Ala His Asp Arg Trp Leu Ala Thr Leu GlnGly Val Asp Ala Val Lys 500 505 510 Ala Val Leu Glu Phe Thr Pro Glu ThrPro Ser Pro Leu Ile Gly Ile 515 520 525 Leu Glu Asn Lys Ile Ile Arg MetPro Leu Val Glu Ser Val Lys Leu 530 535 540 Thr Lys Ser Val Ala Thr AlaIle Glu Asn Lys Asp Phe Asp Lys Ala 545 550 555 560 Ile Ser Leu Arg AspThr Glu Phe Ile Glu Leu Tyr Glu Asn Phe Leu 565 570 575 Ser Thr Thr ValLys Asp Asp Gly Ser Glu Leu Leu Pro Val Ser Asp 580 585 590 Arg Leu AsnIle Gly Ile Val His Val Gly Ala Pro Ser Ala Ala Leu 595 600 605 Asn AlaAla Thr Arg Ala Ala Thr Leu Tyr Cys Leu Ser His Gly His 610 615 620 LysPro Tyr Ala Ile Met Asn Gly Phe Ser Gly Leu Ile Gln Thr Gly 625 630 635640 Glu Val Lys Glu Leu Ser Trp Ile Asp Val Glu Asn Trp His Asn Leu 645650 655 Gly Gly Ser Glu Ile Gly Thr Asn Arg Ser Val Ala Ser Glu Asp Leu660 665 670 Gly Thr Ile Ala Tyr Tyr Phe Gln Lys Asn Lys Leu Asp Gly LeuIle 675 680 685 Ile Leu Gly Gly Phe Glu Gly Phe Arg Ser Leu Lys Gln LeuArg Asp 690 695 700 Gly Arg Thr Gln His Pro Ile Phe Asn Ile Pro Met CysLeu Ile Pro 705 710 715 720 Ala Thr Val Ser Asn Asn Val Pro Gly Thr GluTyr Ser Leu Gly Val 725 730 735 Asp Thr Cys Leu Asn Ala Leu Val Asn TyrThr Asp Asp Ile Lys Gln 740 745 750 Ser Ala Ser Ala Thr Arg Arg Arg ValPhe Val Cys Glu Val Gln Gly 755 760 765 Gly His Ser Gly Tyr Ile Ala SerPhe Thr Gly Leu Ile Thr Gly Ala 770 775 780 Val Ser Val Tyr Thr Pro GluLys Lys Ile Asp Leu Ala Ser Ile Arg 785 790 795 800 Glu Asp Ile Thr LeuLeu Lys Glu Asn Phe Arg His Asp Lys Gly Glu 805 810 815 Asn Arg Asn GlyLys Leu Leu Val Arg Asn Glu Gln Ala Ser Ser Val 820 825 830 Tyr Ser ThrGln Leu Leu Ala Asp Ile Ile Ser Glu Ala Ser Lys Gly 835 840 845 Lys PheGly Val Arg Thr Ala Ile Pro Gly His Val Gln Gln Gly Gly 850 855 860 ValPro Ser Ser Lys Asp Arg Val Thr Ala Ser Arg Phe Ala Val Lys 865 870 875880 Cys Ile Lys Phe Ile Glu Gln Trp Asn Lys Lys Asn Glu Ala Ser Pro 885890 895 Asn Thr Asp Ala Lys Val Leu Arg Phe Lys Phe Asp Thr His Gly Glu900 905 910 Lys Val Pro Thr Val Glu His Glu Asp Asp Ser Ala Ala Val IleCys 915 920 925 Val Asn Gly Ser His Val Ser Phe Lys Pro Ile Ala Asn LeuTrp Glu 930 935 940 Asn Glu Thr Asn Val Glu Leu Arg Lys Gly Phe Glu ValHis Trp Ala 945 950 955 960 Glu Tyr Asn Lys Ile Gly Asp Ile Leu Ser GlyArg Leu Lys Leu Arg 965 970 975 Ala Glu Val Ala Ala Leu Ala Ala Glu AsnLys 980 985 42 2000 DNA Saccharomyces cerevisiae 42 gatcacagtttttcgaggat agcactgatc tatacaataa aaggagctat tttcatcata 60 atgaaagagatgtatttgta ttcattttcg cacttgccac cattttacta attgtttgct 120 catgttatgttacgcctcta tatcgtatgc atcacaagat gggaacttgg aattggtcct 180 cggcgttagattgcgccttc attggtgcct tctcaattga atttatcgtg aaaacagtag 240 ctgacggatttatatattct ccaaatgctt acctgaggaa tccatggaac tttattgatt 300 tttgtgtcctaatctcaatg tggattaatt taattgcata cctaaaaaac aatggaaatt 360 tgtctaggattttcaaggga ttgacagccc tgagggccct cagatgcctc acgatcagta 420 acacagctcgtcaaacattt aacctagtta tgtttgatgg tttaaataaa atttttgaag 480 ctgggttgatttcactcagt ttgctatttc catttacagt ttggggctta agcattttta 540 aaggccgtttaggtacttgc aatgacggaa gtttgggccg tgcagattgt tacaatgaat 600 attcaaattccgtttttcaa tgggatatca tgtctccaag ggtttaccag caaccatatc 660 ttcatttggactctttcgca agcgctttta gttcattata ccaaatcatt tctttggaag 720 gatgggttgatttgttggaa aatatgatga atagttcagg aataggtaca cccgctacgg 780 taatgggttcagcagggaat gctttattcc tcgttctgtt taatttttta agtatggttt 840 tcatcctgaacttgtttgtt tcattcattg ttaacaacca agcaaggaca acaggaagcg 900 cttactttaccattgaggaa aaggcgtggc tggaatccca gaaactttta tctcaggcca 960 agccaaaagctatcccaaat ttaattgagt tatcaagagt taggcaattt ttctatcaac 1020 ttgcagtggagaaaaaaaat ttctactacg catcgtttct tcaggtagta ctttatttgc 1080 acataatcatgctcctgagt cgaagctaca atccaggaaa cttgataggt tatcaaggtg 1140 tttattttatgttttccact agtgtttttt taattcaaga ggcacttcac atgtgcggtg 1200 aaggaccaagattatatttt aggcaaaaat ggaacagcat acgactcagt atcataatta 1260 tagcctttattatgaacgct gtagcattcc acgttccagc ctctcactat tggttccaca 1320 atataaaggggtttttcctg ttagtgatat ttttgtttat tattcctcaa aatgacacac 1380 taactgaactattagaaacc gcaatggcaa gcttaccgcc tattctatca ttgacctaca 1440 cttggggggttttattttta gtatatgcta ttgctttgaa tcaaatcttc ggcctaacaa 1500 ggttagggagtaatacgacc gataacataa attttagaac tgtaatcaaa tccatgattg 1560 ttctgtttagatgtagtttt ggtgagggct ggaattatat catggccgac ctaactgtgt 1620 cagaaccttattgctcctct gatgataatt caacctatac ggactgtgga tcagagacat 1680 atgcctatttgttattaatg tcgtggaata ttatttccat gtatattttt gtgaatatgt 1740 ttgtttcgttgattattggt aatttcagtt atgtttaccg tagcggtgga tctcgctctg 1800 gcatcaacagatcggagata aaaaaataca ttgaagcttg gtccaaattt gatactgatg 1860 gaactggcgagcttgagctg tcctacctcc caagaataat gcattcattt gacggtcctc 1920 tttcatttaaaatttgggaa ggtagattga caataaaaag tctagtcgag aactacatgg 1980 aggttaacccagatgatcca 2000 43 465 PRT Saccharomyces cerevisiae 43 Met Asp Gly HisAsn Gln Asn Gln Tyr Gln Asn Gln Asn Gln Ile Gln 1 5 10 15 Gln Ser GlnGln Pro Pro Leu Lys Lys Tyr Val Thr Gln Arg Arg Ser 20 25 30 Val Asp ValSer Ser Pro Tyr Ile Asn Leu Tyr Tyr Asn Arg Arg His 35 40 45 Gly Leu ProAsn Leu Val Val Glu Pro Glu Thr Ser Tyr Thr Ile Asp 50 55 60 Ile Met ProPro Asn Ala Tyr Arg Gly Arg Asp Arg Val Ile Asn Leu 65 70 75 80 Pro SerLys Phe Thr His Leu Ser Ser Asn Lys Val Lys His Val Ile 85 90 95 Pro AlaIle Gln Trp Thr Pro Glu Gly Arg Arg Leu Val Val Ala Thr 100 105 110 TyrSer Gly Glu Phe Ser Leu Trp Asn Ala Ser Ser Phe Thr Phe Glu 115 120 125Thr Leu Met Gln Ala His Asp Ser Ala Val Thr Thr Met Lys Tyr Ser 130 135140 His Asp Ser Asp Trp Met Ile Ser Gly Asp Ala Asp Gly Met Ile Lys 145150 155 160 Ile Trp Gln Pro Asn Phe Ser Met Val Lys Glu Ile Asp Ala AlaHis 165 170 175 Thr Glu Ser Ile Arg Asp Met Ala Phe Ser Ser Asn Asp SerLys Phe 180 185 190 Val Thr Cys Ser Asp Asp Asn Ile Leu Lys Ile Trp AsnPhe Ser Asn 195 200 205 Gly Lys Gln Glu Arg Val Leu Ser Gly His His TrpAsp Val Lys Ser 210 215 220 Cys Asp Trp His Pro Glu Met Gly Leu Ile AlaSer Ala Ser Lys Asp 225 230 235 240 Asn Leu Val Lys Leu Trp Asp Pro ArgSer Gly Asn Cys Ile Ser Ser 245 250 255 Ile Leu Lys Phe Lys His Thr ValLeu Lys Thr Arg Phe Gln Pro Thr 260 265 270 Lys Gly Asn Leu Leu Met AlaIle Ser Lys Asp Lys Ser Cys Arg Val 275 280 285 Phe Asp Ile Arg Tyr SerMet Lys Glu Leu Met Cys Val Arg Asp Glu 290 295 300 Thr Asp Tyr Met ThrLeu Glu Trp His Pro Ile Asn Glu Ser Met Phe 305 310 315 320 Thr Leu AlaCys Tyr Asp Gly Ser Leu Lys His Phe Asp Leu Leu Gln 325 330 335 Asn LeuAsn Glu Pro Ile Leu Thr Ile Pro Tyr Ala His Asp Lys Cys 340 345 350 IleThr Ser Leu Ser Tyr Asn Pro Val Gly His Ile Phe Ala Thr Ala 355 360 365Ala Lys Asp Arg Thr Ile Arg Phe Trp Thr Arg Ala Arg Pro Ile Asp 370 375380 Pro Asn Ala Tyr Asp Asp Pro Thr Tyr Asn Asn Lys Lys Ile Asn Gly 385390 395 400 Trp Phe Phe Gly Ile Asn Asn Asp Ile Asn Ala Val Arg Glu LysSer 405 410 415 Glu Phe Gly Ala Ala Pro Pro Pro Pro Ala Thr Leu Glu ProHis Ala 420 425 430 Leu Pro Asn Met Asn Gly Phe Ile Asn Lys Lys Pro ArgGln Glu Ile 435 440 445 Pro Gly Ile Asp Ser Asn Ile Lys Ser Ser Thr LeuPro Gly Leu Ser 450 455 460 Ile 465 44 1398 DNA Saccharomyces cerevisiae44 atggacgggc ataatcagaa ccagtaccag aatcagaatc aaattcagca gtcacaacaa 60ccacctctga agaaatatgt gacacagaga cgatcggtag acgtgagttc tccgtatatt 120aacctatatt acaacagacg acatggatta ccgaacctag tggtagagcc agaaacttca 180tacacaatag atataatgcc gcctaatgcc tacagaggtc gagatcgagt cataaatttg 240cccagcaaat ttacgcattt aagctcgaat aaagtgaaac atgtgatacc cgccatccaa 300tggaccccag aaggcagaag acttgtagta gcaacttata gcggagaatt ttctctatgg 360aacgcatctt cgtttacgtt tgaaaccctc atgcaagcac atgattcggc tgtgactaca 420atgaaatatt ctcacgacag tgattggatg attagtgggg acgcagatgg aatgattaag 480atatggcaac caaactttag tatggttaaa gaaattgatg ctgcacacac ggaaagtatt 540agggatatgg cattcagtag taacgactcg aaatttgtta cctgttcaga tgataatatc 600ttgaagattt ggaacttcag caatggcaaa caagaaagag tattatcggg gcaccactgg 660gacgtgaaaa gttgtgattg gcatcccgag atggggttaa ttgcctccgc atctaaggac 720aatttagtaa aactgtggga tccgcgttcc ggaaactgca tatcatcaat tctgaagttc 780aaacatactg tcttgaagac gcgatttcaa cccaccaagg gaaatctatt aatggcgatt 840tcaaaagata aatcgtgtcg tgtgttcgac attagataca gtatgaagga actaatgtgt 900gtaagagatg aaacggatta tatgacacta gaatggcacc caataaatga atctatgttc 960actctggcgt gctatgacgg ctcgctaaag cattttgatc ttttacaaaa tctaaacgaa 1020cctattttga ctattccata cgcccatgat aagtgtatca cctcgttatc atataatcca 1080gtaggacata tatttgccac agcagctaag gatagaacca taagattttg gactagagcg 1140aggccaattg atcctaatgc atatgatgat ccaacttata acaataagaa gataaatgga 1200tggttttttg gcatcaataa cgacatcaat gctgtgagag agaagagtga attcggtgcg 1260gccccgccac caccagctac acttgaacca catgcgttac caaacatgaa tggattcatc 1320aacaagaaac cgcgtcaaga aatcccaggc atcgattcaa acatcaagag ttccacattg 1380ccaggtttaa gcatataa 1398 45 785 PRT Saccharomyces cerevisiae 45 Met SerSer Ala Glu Met Glu Gln Leu Leu Gln Ala Lys Thr Leu Ala 1 5 10 15 MetHis Asn Asn Pro Thr Glu Met Leu Pro Lys Val Leu Glu Thr Thr 20 25 30 AlaSer Met Tyr His Asn Gly Asn Leu Ser Lys Leu Lys Leu Pro Leu 35 40 45 AlaLys Phe Phe Thr Gln Leu Val Leu Asp Val Val Ser Met Asp Ser 50 55 60 ProIle Ala Asn Thr Glu Arg Pro Phe Ile Ala Ala Gln Tyr Leu Pro 65 70 75 80Leu Leu Leu Ala Met Ala Gln Ser Thr Ala Asp Val Leu Val Tyr Lys 85 90 95Asn Ile Val Leu Ile Met Cys Ala Ser Tyr Pro Leu Val Leu Asp Leu 100 105110 Val Ala Lys Thr Ser Asn Gln Glu Met Phe Asp Gln Leu Cys Met Leu 115120 125 Lys Lys Phe Val Leu Ser His Trp Arg Thr Ala Tyr Pro Leu Arg Ala130 135 140 Thr Val Asp Asp Glu Thr Asp Val Glu Gln Trp Leu Ala Gln IleAsp 145 150 155 160 Gln Asn Ile Gly Val Lys Leu Ala Thr Ile Lys Phe IleSer Glu Val 165 170 175 Val Leu Ser Gln Thr Lys Ser Pro Ser Gly Asn GluIle Asn Ser Ser 180 185 190 Thr Ile Pro Asp Asn His Pro Val Leu Asn LysPro Ala Leu Glu Ser 195 200 205 Glu Ala Lys Arg Leu Leu Asp Met Leu LeuAsn Tyr Leu Ile Glu Glu 210 215 220 Gln Tyr Met Val Ser Ser Val Phe IleGly Ile Ile Asn Ser Leu Ser 225 230 235 240 Phe Val Ile Lys Arg Arg ProGln Thr Thr Ile Arg Ile Leu Ser Gly 245 250 255 Leu Leu Arg Phe Asn ValAsp Ala Lys Phe Pro Leu Glu Gly Lys Ser 260 265 270 Asp Leu Asn Tyr LysLeu Ser Lys Arg Phe Val Glu Arg Ala Tyr Lys 275 280 285 Asn Phe Val GlnPhe Gly Leu Lys Asn Gln Ile Ile Thr Lys Ser Leu 290 295 300 Ser Ser GlySer Gly Ser Ser Ile Tyr Ser Lys Leu Thr Lys Ile Ser 305 310 315 320 GlnThr Leu His Val Ile Gly Glu Glu Thr Lys Ser Lys Gly Ile Leu 325 330 335Asn Phe Asp Pro Ser Lys Gly Asn Ser Lys Lys Thr Leu Ser Arg Gln 340 345350 Asp Lys Leu Lys Tyr Ile Ser Leu Trp Lys Arg Gln Leu Ser Ala Leu 355360 365 Leu Ser Thr Leu Gly Val Ser Thr Lys Thr Pro Thr Pro Val Ser Ala370 375 380 Pro Ala Thr Gly Ser Ser Thr Glu Asn Met Leu Asp Gln Leu LysIle 385 390 395 400 Leu Gln Lys Tyr Thr Leu Asn Lys Ala Ser His Gln GlyAsn Thr Phe 405 410 415 Phe Asn Asn Ser Pro Lys Pro Ile Ser Asn Thr TyrSer Ser Val Tyr 420 425 430 Ser Leu Met Asn Ser Ser Asn Ser Asn Gln AspVal Thr Gln Leu Pro 435 440 445 Asn Asp Ile Leu Ile Lys Leu Ser Thr GluAla Ile Leu Gln Met Asp 450 455 460 Ser Thr Lys Leu Ile Thr Gly Leu SerIle Val Ala Ser Arg Tyr Thr 465 470 475 480 Asp Leu Met Asn Thr Tyr IleAsn Ser Val Pro Ser Ser Ser Ser Ser 485 490 495 Lys Arg Lys Ser Asp AspAsp Asp Asp Gly Asn Asp Asn Glu Glu Val 500 505 510 Gly Asn Asp Gly ProThr Ala Asn Ser Lys Lys Ile Lys Met Glu Thr 515 520 525 Glu Pro Leu AlaGlu Glu Pro Glu Glu Pro Glu Asp Asp Asp Arg Met 530 535 540 Gln Lys MetLeu Gln Glu Glu Glu Ser Ala Gln Glu Ile Ser Gly Asp 545 550 555 560 AlaAsn Lys Ser Thr Ser Ala Ile Lys Glu Ile Ala Pro Pro Phe Glu 565 570 575Pro Asp Ser Leu Thr Gln Asp Glu Lys Leu Lys Tyr Leu Ser Lys Leu 580 585590 Thr Lys Lys Leu Phe Glu Leu Ser Gly Arg Gln Asp Thr Thr Arg Ala 595600 605 Lys Ser Ser Ser Ser Ser Ser Ile Leu Leu Asp Asp Asp Asp Ser Ser610 615 620 Ser Trp Leu His Val Leu Ile Arg Leu Val Thr Arg Gly Ile GluAla 625 630 635 640 Gln Glu Ala Ser Asp Leu Ile Arg Glu Glu Leu Leu GlyPhe Phe Ile 645 650 655 Gln Asp Phe Glu Gln Arg Val Ser Leu Ile Ile GluTrp Leu Asn Glu 660 665 670 Glu Trp Phe Phe Gln Thr Ser Leu His Gln AspPro Ser Asn Tyr Lys 675 680 685 Lys Trp Ser Leu Arg Val Leu Glu Ser LeuGly Pro Phe Leu Glu Asn 690 695 700 Lys His Arg Arg Phe Phe Ile Arg LeuMet Ser Glu Leu Pro Ser Leu 705 710 715 720 Gln Ser Asp His Leu Glu AlaLeu Lys Pro Ile Cys Leu Asp Pro Ala 725 730 735 Arg Ser Ser Leu Gly PheGln Thr Leu Lys Phe Leu Ile Met Phe Arg 740 745 750 Pro Pro Val Gln AspThr Val Arg Asp Leu Leu His Gln Leu Lys Gln 755 760 765 Glu Asp Glu GlyLeu His Lys Gln Cys Asp Ser Leu Leu Asp Arg Leu 770 775 780 Lys 785 462000 DNA Saccharomyces cerevisiae 46 atgtcatctg cagagatgga acaattgttacaggccaaga cactggccat gcacaacaat 60 ccaacggaga tgctgcccaa ggtgctcgaaactacggcat ccatgtacca caacggtaat 120 ctcagcaagc tgaagttgcc tttggccaagttttttacac agttagttct agacgtggtg 180 tcgatggact ctccaattgc gaatactgagagaccgttta ttgctgctca atatctgcca 240 ctacttcttg ctatggcgca atccaccgcggacgtactag tgtacaagaa tatcgtgctt 300 attatgtgcg cttcataccc gctggtgttggatctggttg ctaagacatc aaaccaggaa 360 atgtttgatc agttgtgtat gctgaagaagttcgtgctct cgcactggag aactgcatat 420 cctttgcgtg ccaccgttga cgatgaaacggatgtcgaac aatggctggc gcagattgac 480 caaaatatcg gcgtgaaatt agcgaccatcaagttcatat ctgaggtcgt gctgtcgcaa 540 actaaatcac ccagcggcaa cgagattaattcatctacca tcccggataa ccaccctgtg 600 ttgaacaaac cggctttgga gagcgaggctaagaggcttc ttgatatgtt gctaaactac 660 ctaattgagg aacagtacat ggtctcgtccgttttcattg gtatcatcaa ttctttatcc 720 ttcgtcatca aaagaaggcc gcagacaacaataagaattc tttccgggct gttgcgtttc 780 aacgtcgacg ccaagtttcc cctagagggcaagtctgact tgaactacaa actatccaag 840 agatttgttg aaagggcgta caagaactttgtgcaatttg ggctaaaaaa tcaaatcatt 900 acaaaatccc tctcatccgg atcagggtcatcgatctact ccaagctgac caagatttct 960 caaactttac acgttattgg cgaagagaccaagagcaagg gaattttgaa cttcgaccct 1020 tccaagggca atagcaagaa aacgttgtccaggcaggaca aactaaaata catctcacta 1080 tggaaaaggc aattatccgc gttattgtctactctagggg tgtccacaaa gacccccacg 1140 cctgtgtccg cacctgcaac gggctcttcaaccgaaaaca tgcttgatca actgaagata 1200 ttgcaaaaat acaccctcaa caaggcttcacaccagggca atactttttt caacaactca 1260 cccaaaccaa tcagcaacac ctactcatctgtgtactcat tgatgaacag ttcgaactcc 1320 aaccaggatg tgacccagct acccaatgacatacttatca agctgtccac agaggccatc 1380 ttgcaaatgg acagcacgaa actgatcaccggattgtcta tcgttgcttc gaggtacacg 1440 gatttaatga atacgtacat caattctgtaccgtcctcgt catcatcaaa gaggaaatcc 1500 gacgatgatg acgacggcaa cgacaatgaagaagttggaa acgatggccc aacggctaat 1560 agcaagaaaa tcaaaatgga aacagaaccactagcggagg aaccagagga gcccgaagac 1620 gatgaccgaa tgcagaagat gcttcaagaagaggaaagcg cccaagaaat ctcaggagat 1680 gccaacaaat caacttctgc cattaaggagatcgcacccc cctttgaacc tgactcattg 1740 acgcaggatg aaaaactaaa gtacctctcaaagctgacca agaaactgtt tgaattatcc 1800 ggtcgccagg atactacccg ggccaaatcttcgtcttcct cctccatatt actggacgat 1860 gacgactcct cgtcatggtt acacgtcttaatcagattgg ttacgagagg aatcgaagca 1920 caagaggcca gtgacctgat tcgtgaagaactgcttggct tcttcatcca ggatttcgag 1980 caacgtgtca gtctgatcat 2000 47 533PRT Saccharomyces cerevisiae 47 Met Ser Ala Pro Val Pro Gln Leu Val AsnIle Ser His Ala Leu Gln 1 5 10 15 Ala Ser Thr Ile Gln Gln Ile Arg LeuAsp Met Val Asp Phe Asn Lys 20 25 30 Asp Cys Lys Leu Ser Ser Ile Gln LeuAla Arg Ile Asp Lys Tyr Ile 35 40 45 Asp Ser Leu Gln Ala Ala Leu Asn GlnPhe Thr Lys Asp Asn Leu His 50 55 60 Ile Glu Arg Lys Glu Lys Asn Val ThrGlu Ala Asp Ile Gln Leu Tyr 65 70 75 80 Ser Gly Leu Lys Ser Met Tyr LeuAsp Tyr Leu Asn Gln Leu Ile Lys 85 90 95 Leu Lys His Glu Lys Gln His HisSer Thr Pro Pro Ile Ala Asn Asp 100 105 110 Val Ser Leu Asp Phe Phe ValAsn Gln Leu Pro Lys Phe Ser Pro Glu 115 120 125 Glu Arg Lys Asn Tyr IleAsp Asn Leu Ile Leu Asn Lys Asn Ser His 130 135 140 Asn Arg Leu Ser LysMet Asp Gly Leu Val Asp Ala Val Ile Asn Leu 145 150 155 160 Cys Val LeuAsp Thr Ser Val Ala Glu Asn Val Arg Ser Tyr Met Lys 165 170 175 Leu LeuAsp Thr Leu Gly Phe Gln Lys Gly Ser Asn Ser Thr Gly Thr 180 185 190 LysAla Asn Leu Lys Lys Lys Leu Ala Ser Ser Lys Ala Lys Ile Lys 195 200 205Asp Ser Glu Lys Glu Lys Glu Lys Glu Lys Asp Lys Ser Lys Val Lys 210 215220 Met Lys Thr Lys Leu Lys Pro Ser Pro Leu Leu Asn Asn Asp Asp Lys 225230 235 240 Asn Ser Ser Pro Ser Pro Thr Ala Ser Thr Ser Ser Met Lys LysLeu 245 250 255 Lys Ser Gly Leu Phe Asn Lys Asn Glu Ala Lys Ser Thr GluSer Leu 260 265 270 Pro Thr Ser Ser Lys Lys Lys Leu Ser Phe Ser Lys TyrLeu Asn Lys 275 280 285 Asp Asp Ala Asp Met Thr Lys Leu Gly Thr Lys ArgSer Ile Asp Val 290 295 300 Asp Phe Lys Val Asn Pro Glu Ala Ser Thr ValAla Ser Asn Ile Ile 305 310 315 320 Ser Ser Ser Thr Ser Gly Ser Ser ThrThr Thr Val Ala Thr Pro Ala 325 330 335 Ser Ser Glu Glu Pro Leu Lys LysLys Thr Lys Ile Ser Val Gln Asp 340 345 350 Ser Asn Val Gln Ser Ile LeuArg Asn Gly Lys Pro Lys Lys Ala Arg 355 360 365 Ile Ser Ser Ile Lys PheLeu Asp Asp Ser Gln Leu Ile Lys Val Tyr 370 375 380 Gly Asp Asp Leu ProAsn Gln Gly Leu Gln Val Ser Pro Thr Gln Leu 385 390 395 400 Lys Lys IleLeu Lys Pro Phe Lys Glu Gly Glu Pro Lys Glu Ile Ile 405 410 415 Leu PheGlu Asp Met Ser Ile Lys Leu Lys Pro Leu Asp Leu Met Phe 420 425 430 LeuLys Asn Thr Asn Ser Asp Asp Tyr Met Asp Ile Ser Glu Thr Lys 435 440 445Gly Gly Pro Ile His Cys Glu Thr Arg Thr Pro Leu Ile Tyr Arg Lys 450 455460 Asn Phe Asn His Phe Asn Pro Asp Leu Asn Lys Arg Pro Pro Arg Glu 465470 475 480 Pro Ile Glu Phe Asp Leu Asn Gly Asn Thr Asn Ser Thr Pro ThrIle 485 490 495 Ala Lys Ala Phe Gly Lys Asn Ser Leu Leu Leu Arg Lys AspArg Gly 500 505 510 Gly Leu Pro Tyr Lys His Val Pro Ile Val Lys Arg AsnLys Tyr Pro 515 520 525 Pro Arg Pro Val His 530 48 1602 DNASaccharomyces cerevisiae 48 atgtcagcac ctgttcctca attagtaaat atttctcatgccttgcaagc ttccactatc 60 cagcaaattc gtttggatat ggtagatttt aataaagattgcaaattatc ttccattcaa 120 ctggcaagaa ttgacaagta tatcgattct ttgcaagcggctttgaatca gtttaccaaa 180 gataacttgc acatagaacg gaaggaaaag aacgtgactgaagcagatat acagctgtat 240 tctggcttaa aatcaatgta tctagattat ttgaaccagctaattaaact aaagcatgag 300 aaacaacacc actctacacc gcccatcgct aatgatgtctccctggactt cttcgttaac 360 caattgccta agttttctcc cgaggaaagg aaaaattacattgacaattt aatcctgaat 420 aaaaatagcc ataatcgatt atctaaaatg gatggtctggtagatgcagt catcaattta 480 tgcgtcttgg atacttccgt tgctgaaaat gtacgatcctatatgaaatt attagataca 540 ttgggtttcc aaaagggttc gaatagtact ggtacaaaggccaacctcaa aaagaaattg 600 gctagttcga aagcaaagat aaaggattca gaaaaagaaaaggagaagga gaaggataaa 660 tcaaaagtca agatgaaaac taaattaaaa ccttctcctttgctcaataa cgatgacaaa 720 aattcttctc catcgcctac tgcatctacc tcttcaatgaagaaattgaa gtcgggttta 780 ttcaataaaa atgaagctaa gtctacagaa tctctacctacttcttccaa gaaaaaacta 840 tcattttcta aatatctgaa caaggatgac gcagatatgaccaagcttgg gactaaacgg 900 tcaatagatg tggatttcaa agtcaacccc gaagcatccacggtggcttc taatatcata 960 tcttcgtcaa cgtcaggatc gtcaaccaca acggtagcgactcctgcttc ttcagaagag 1020 cccttaaaaa aaaaaaccaa aatatccgtg caagactctaatgtacaatc gattttgaga 1080 aatggtaaac cgaaaaaagc acgcataagt agcatcaaatttttggatga ttcccaacta 1140 ataaaagttt acggtgacga tctaccgaac caagggctacaagtttctcc tactcaattg 1200 aaaaaaattc tgaaaccatt caaggagggg gaaccgaaggaaattatatt gttcgaggat 1260 atgtcaatca aattaaaacc tcttgatttg atgtttctgaagaacacaaa cagtgatgac 1320 tatatggata tatccgagac taaaggtggc ccaatacattgtgaaacaag gaccccgttg 1380 atctatagaa aaaatttcaa tcatttcaac ccggacttgaataaaaggcc gccaagagaa 1440 cccatagaat tcgacttaaa tggaaatacg aactcaaccccgactatagc aaaggctttc 1500 ggtaaaaata gtttattact aaggaaggac agaggtggtttgccatacaa gcatgtcccc 1560 atagtaaaaa gaaataaata tcctccaaga ccagtacactaa 1602 49 677 PRT Saccharomyces cerevisiae 49 Met Ser Ser Ser Thr ThrPro Asp Leu Leu Tyr Pro Ser Ala Asp Lys 1 5 10 15 Val Ala Glu Pro SerAsp Asn Ile His Gly Asp Glu Leu Arg Leu Arg 20 25 30 Glu Arg Ile Lys AspAsn Pro Thr Asn Ile Leu Ser Tyr Phe Gln Leu 35 40 45 Ile Gln Tyr Leu GluThr Gln Glu Ser Tyr Ala Lys Val Arg Glu Val 50 55 60 Tyr Glu Gln Phe HisAsn Thr Phe Pro Phe Tyr Ser Pro Ala Trp Thr 65 70 75 80 Leu Gln Leu LysGly Glu Leu Ala Arg Asp Glu Phe Glu Thr Val Glu 85 90 95 Lys Ile Leu AlaGln Cys Leu Ser Gly Lys Leu Glu Asn Asn Asp Leu 100 105 110 Ser Leu TrpSer Thr Tyr Leu Asp Tyr Ile Arg Arg Lys Asn Asn Leu 115 120 125 Ile ThrGly Gly Gln Glu Ala Arg Ala Val Ile Val Lys Ala Phe Gln 130 135 140 LeuVal Met Gln Lys Cys Ala Ile Phe Glu Pro Lys Ser Ser Ser Phe 145 150 155160 Trp Asn Glu Tyr Leu Asn Phe Leu Glu Gln Trp Lys Pro Phe Asn Lys 165170 175 Trp Glu Glu Gln Gln Arg Ile Asp Met Leu Arg Glu Phe Tyr Lys Lys180 185 190 Met Leu Cys Val Pro Phe Asp Asn Leu Glu Lys Met Trp Asn ArgTyr 195 200 205 Thr Gln Trp Glu Gln Glu Ile Asn Ser Leu Thr Ala Arg LysPhe Ile 210 215 220 Gly Glu Leu Ser Ala Glu Tyr Met Lys Ala Arg Ser LeuTyr Gln Glu 225 230 235 240 Trp Leu Asn Val Thr Asn Gly Leu Lys Arg AlaSer Pro Ile Asn Leu 245 250 255 Arg Thr Ala Asn Lys Lys Asn Ile Pro GlnPro Gly Thr Ser Asp Ser 260 265 270 Asn Ile Gln Gln Leu Gln Ile Trp LeuAsn Trp Ile Lys Trp Glu Arg 275 280 285 Glu Asn Lys Leu Met Leu Ser GluAsp Met Leu Ser Gln Arg Ile Ser 290 295 300 Tyr Val Tyr Lys Gln Gly IleGln Tyr Met Ile Phe Ser Ala Glu Met 305 310 315 320 Trp Tyr Asp Tyr SerMet Tyr Ile Ser Glu Asn Ser Asp Arg Gln Asn 325 330 335 Ile Leu Tyr ThrAla Leu Leu Ala Asn Pro Asp Ser Pro Ser Leu Thr 340 345 350 Phe Lys LeuSer Glu Cys Tyr Glu Leu Asp Asn Asp Ser Glu Ser Val 355 360 365 Ser AsnCys Phe Asp Lys Cys Thr Gln Thr Leu Leu Ser Gln Tyr Lys 370 375 380 LysIle Ala Ser Asp Val Asn Ser Gly Glu Asp Asn Asn Thr Glu Tyr 385 390 395400 Glu Gln Glu Leu Leu Tyr Lys Gln Arg Glu Lys Leu Thr Phe Val Phe 405410 415 Cys Val Tyr Met Asn Thr Met Lys Arg Ile Ser Gly Leu Ser Ala Ala420 425 430 Arg Thr Val Phe Gly Lys Cys Arg Lys Leu Lys Arg Ile Leu ThrHis 435 440 445 Asp Val Tyr Val Glu Asn Ala Tyr Leu Glu Phe Gln Asn GlnAsn Asp 450 455 460 Tyr Lys Thr Ala Phe Lys Val Leu Glu Leu Gly Leu LysTyr Phe Gln 465 470 475 480 Asn Asp Gly Val Tyr Ile Asn Lys Tyr Leu AspPhe Leu Ile Phe Leu 485 490 495 Asn Lys Asp Ser Gln Ile Lys Thr Leu PheGlu Thr Ser Val Glu Lys 500 505 510 Val Gln Asp Leu Thr Gln Leu Lys GluIle Tyr Lys Lys Met Ile Ser 515 520 525 Tyr Glu Ser Lys Phe Gly Asn LeuAsn Asn Val Tyr Ser Leu Glu Lys 530 535 540 Arg Phe Phe Glu Arg Phe ProGln Glu Asn Leu Ile Glu Val Phe Thr 545 550 555 560 Ser Arg Tyr Gln IleGln Asn Ser Asn Leu Ile Lys Lys Leu Glu Leu 565 570 575 Thr Tyr Met TyrAsn Glu Glu Glu Asp Ser Tyr Phe Ser Ser Gly Asn 580 585 590 Gly Asp GlyHis His Gly Ser Tyr Asn Met Ser Ser Ser Asp Arg Lys 595 600 605 Arg LeuMet Glu Glu Thr Gly Asn Asn Gly Asn Phe Ser Asn Lys Lys 610 615 620 PheLys Arg Asp Ser Glu Leu Pro Thr Glu Val Leu Asp Leu Leu Ser 625 630 635640 Val Ile Pro Lys Arg Gln Tyr Phe Asn Thr Asn Leu Leu Asp Ala Gln 645650 655 Lys Leu Val Asn Phe Leu Asn Asp Gln Val Glu Ile Pro Thr Val Glu660 665 670 Ser Thr Lys Ser Gly 675 50 2000 DNA Saccharomyces cerevisiae50 atgtccagct ctacgactcc tgatttacta tatccctctg cggacaaagt cgcagagcct 60agtgacaata tacatggaga tgaactacga cttagagaaa ggattaaaga caatcccacg 120aatattttat catacttcca gcttattcaa tatttggaaa ctcaagagtc atacgctaag 180gtgagagaag tatacgagca atttcataac acattcccgt tttattcacc tgcgtggact 240ttgcaactaa agggtgaatt ggcaagagat gaatttgaga ctgttgagaa gattttggct 300caatgtcttt ctggcaagtt ggaaaataat gacctatctc tttggtcaac atatttggac 360tacatacgca gaaaaaacaa cttaattact ggtggacaag aggcgagagc tgttattgtc 420aaggcattcc aactagttat gcaaaagtgt gcaatttttg aacccaaatc atcttctttt 480tggaacgaat atctcaattt tttagagcag tggaagccat tcaacaaatg ggaggagcaa 540cagcgaattg acatgctcag agaattctac aagaaaatgc tatgtgttcc ttttgataat 600ctagaaaaaa tgtggaatag atacactcaa tgggaacaag aaataaattc cctaacagcc 660agaaaattta ttggcgagtt atcagccgaa tacatgaaag cccgttcctt ataccaggaa 720tggttgaacg ttactaatgg attgaaaagg gcatctccaa ttaatctgcg cacagcaaac 780aagaaaaaca taccacaacc aggtacctca gactcaaaca ttcagcagtt acagatttgg 840ttgaattgga taaaatggga aagggagaat aagttgatgc ttagtgaaga tatgctatca 900caaagaatca gttacgttta taaacaaggt attcaataca tgatattttc tgctgaaatg 960tggtacgatt attcaatgta tatatctgaa aattcggatc gacaaaatat cttatatact 1020gcgttattag ctaatcccga ctcaccttct cttacattca agttatccga atgctacgaa 1080ctggataatg attctgaaag tgtttctaac tgttttgaca agtgcactca aactttacta 1140tcgcagtata aaaagatcgc ctccgatgta aattcgggtg aagataataa cacagagtat 1200gaacaagagc tgctatacaa acagagggaa aaattaacat tcgtgttttg cgtgtatatg 1260aatacgatga aaagaatatc aggactatcc gcagcacgta ctgtatttgg taaatgtcgt 1320aaactgaagc gtatattaac acatgacgtc tacgtggaaa atgcatattt agaatttcaa 1380aatcaaaacg attataagac tgcttttaag gttttagaat tgggtttaaa atacttccaa 1440aacgatggag tttatatcaa caaatactta gattttttaa tatttttaaa taaggattcg 1500cagatcaaaa ccttatttga aacatcagtg gaaaaagtgc aagatttaac ccagctgaag 1560gaaatataca agaaaatgat aagttatgaa tcgaaattcg gtaacttaaa caacgtttat 1620tctctagaga aaagattttt cgaacggttc ccccaagaaa atttgattga agttttcaca 1680agtcgttatc aaattcaaaa ctccaactta ataaagaaat tagagttaac ttatatgtat 1740aatgaggaag aagacagtta cttttcttct ggaaacgggg atggccatca tggctcttac 1800aatatgagtt cgtcagatag aaagagacta atggaggaaa ctggaaacaa tggaaacttc 1860tccaataaga aattcaaaag agactcagag cttccaacag aggttcttga tttattgagc 1920gttataccaa aacgtcaata ttttaataca aatttactcg atgcgcagaa attggtgaat 1980tttttaaatg atcaagtaga 2000 51 296 PRT Saccharomyces cerevisiae 51 MetAsn Arg Gln Ser Gly Val Asn Ala Gly Val Gln Asn Asn Pro Pro 1 5 10 15Ser Arg Val Val Tyr Leu Gly Ser Ile Pro Tyr Asp Gln Thr Glu Glu 20 25 30Gln Ile Leu Asp Leu Cys Ser Asn Val Gly Pro Val Ile Asn Leu Lys 35 40 45Met Met Phe Asp Pro Gln Thr Gly Arg Ser Lys Gly Tyr Ala Phe Ile 50 55 60Glu Phe Arg Asp Leu Glu Ser Ser Ala Ser Ala Val Arg Asn Leu Asn 65 70 7580 Gly Tyr Gln Leu Gly Ser Arg Phe Leu Lys Cys Gly Tyr Ser Ser Asn 85 9095 Ser Asp Ile Ser Gly Val Ser Gln Gln Gln Gln Gln Gln Tyr Asn Asn 100105 110 Ile Asn Gly Asn Asn Asn Asn Asn Gly Asn Asn Asn Asn Asn Ser Asn115 120 125 Gly Pro Asp Phe Gln Asn Ser Gly Asn Ala Asn Phe Leu Ser GlnLys 130 135 140 Phe Pro Glu Leu Pro Ser Gly Ile Asp Val Asn Ile Asn MetThr Thr 145 150 155 160 Pro Ala Met Met Ile Ser Ser Glu Leu Ala Lys LysPro Lys Glu Val 165 170 175 Gln Leu Lys Phe Leu Gln Lys Phe Gln Glu TrpThr Arg Ala His Pro 180 185 190 Glu Asp Ala Val Ser Leu Leu Glu Leu CysPro Gln Leu Ser Phe Val 195 200 205 Thr Ala Glu Leu Leu Leu Thr Asn GlyIle Cys Lys Val Asp Asp Leu 210 215 220 Ile Pro Leu Ala Ser Arg Pro GlnGlu Glu Ala Ser Ala Thr Asn Asn 225 230 235 240 Asn Ser Val Asn Glu ValVal Asp Pro Ala Val Leu Asn Lys Gln Lys 245 250 255 Glu Leu Leu Lys GlnVal Leu Gln Leu Asn Asp Ser Gln Ile Ser Ile 260 265 270 Leu Pro Asp AspGlu Arg Met Ala Ile Trp Asp Leu Lys Gln Lys Ala 275 280 285 Leu Arg GlyGlu Phe Gly Ala Phe 290 295 52 891 DNA Saccharomyces cerevisiae 52atgaataggc agagcggtgt gaatgcgggg gtacagaaca acccaccatc ccgagtggtg 60tatctgggtt ctataccata cgatcaaaca gaggagcaga tacttgattt atgtagtaat 120gttgggcccg tgatcaattt gaaaatgatg ttcgaccccc aaactggtag gtcgaaaggg 180tacgcgttta ttgaatttag agatttagag tccagtgcca gcgcagtacg taatttgaat 240ggataccaat taggctctag gtttttgaaa tgcggttact ccagcaatag tgatatatcg 300ggagtttcac aacagcaaca acaacagtac aacaacatta atgggaacaa taacaacaat 360ggaaataata ataataatag taatgggccg gactttcaaa acagcggaaa tgccaatttt 420ctaagtcaaa agtttccaga attgccctct ggtatcgacg ttaacataaa catgaccacc 480cctgctatga tgatatcgag cgaactagct aaaaaaccga aagaggtgca gttgaaattt 540ttacaaaaat tccaagaatg gacaagagcg catcctgaag atgctgtttc gctattagag 600ctgtgtccac agttgagttt tgttacggct gaattattgc taacgaatgg gatatgtaaa 660gtggatgatt tgatcccgtt agcttccagg ccgcaagaag aggcatcggc tacgaataac 720aatagcgtga acgaggtggt ggatccagct gtgcttaaca aacagaaaga actactgaaa 780caggtgttac aactgaatga cagtcaaatt tctatcttgc ccgatgatga aaggatggct 840atttgggact taaaacaaaa agcattaagg ggagaatttg gtgcattttg a 891 53 297 PRTSaccharomyces cerevisiae 53 Met Val Val Ile Ala Asn Ala His Asn Glu LeuIle His Asp Ala Val 1 5 10 15 Leu Asp Tyr Tyr Gly Lys Arg Leu Ala ThrCys Ser Ser Asp Lys Thr 20 25 30 Ile Lys Ile Phe Glu Val Glu Gly Glu ThrHis Lys Leu Ile Asp Thr 35 40 45 Leu Thr Gly His Glu Gly Pro Val Trp ArgVal Asp Trp Ala His Pro 50 55 60 Lys Phe Gly Thr Ile Leu Ala Ser Cys SerTyr Asp Gly Lys Val Leu 65 70 75 80 Ile Trp Lys Glu Glu Asn Gly Arg TrpSer Gln Ile Ala Val His Ala 85 90 95 Val His Ser Ala Ser Val Asn Ser ValGln Trp Ala Pro His Glu Tyr 100 105 110 Gly Pro Leu Leu Leu Val Ala SerSer Asp Gly Lys Val Ser Val Val 115 120 125 Glu Phe Lys Glu Asn Gly ThrThr Ser Pro Ile Ile Ile Asp Ala His 130 135 140 Ala Ile Gly Val Asn SerAla Ser Trp Ala Pro Ala Thr Ile Glu Glu 145 150 155 160 Asp Gly Glu HisAsn Gly Thr Lys Glu Ser Arg Lys Phe Val Thr Gly 165 170 175 Gly Ala AspAsn Leu Val Lys Ile Trp Lys Tyr Asn Ser Asp Ala Gln 180 185 190 Thr TyrVal Leu Glu Ser Thr Leu Glu Gly His Ser Asp Trp Val Arg 195 200 205 AspVal Ala Trp Ser Pro Thr Val Leu Leu Arg Ser Tyr Leu Ala Ser 210 215 220Val Ser Gln Asp Arg Thr Cys Ile Ile Trp Thr Gln Asp Asn Glu Gln 225 230235 240 Gly Pro Trp Lys Lys Thr Leu Leu Lys Glu Glu Lys Phe Pro Asp Val245 250 255 Leu Trp Arg Ala Ser Trp Ser Leu Ser Gly Asn Val Leu Ala LeuSer 260 265 270 Gly Gly Asp Asn Lys Val Thr Leu Trp Lys Glu Asn Leu GluGly Lys 275 280 285 Trp Glu Pro Ala Gly Glu Val His Gln 290 295 54 894DNA Saccharomyces cerevisiae 54 atggtcgtca tagctaatgc gcacaacgaattaatccatg acgctgttct agactattat 60 gggaagcgcc ttgcaacctg ctcttctgacaagacaatca agatctttga agtcgaagga 120 gaaacacaca agttaataga cacgttgactggccacgaag gcccagtttg gcgtgttgat 180 tgggcacatc ctaaattcgg aaccattttggcatcgtgtt cttatgatgg taaagtgttg 240 atttggaagg aagaaaacgg tagatggtctcaaattgccg ttcatgctgt ccactctgct 300 tctgtcaact ctgttcaatg ggctcctcatgaatatggcc ccctactgct ggttgcttcc 360 tctgatggta aggtctccgt agtagagttcaaagaaaacg gtactacttc cccaataatc 420 atcgatgctc atgccattgg cgttaactctgcttcttggg ctccagctac catcgaagaa 480 gatggtgaac acaacggtac taaagaatctcgcaagtttg ttactggggg tgctgacaat 540 ttggtaaaga tttggaagta caattcagatgcccaaactt atgttctgga aagcacctta 600 gaaggtcaca gcgattgggt tagagacgtagcatggtcac ctactgttct tctacgttct 660 tatttggcca gtgtttctca agatcgcacctgtattattt ggactcaaga caatgaacaa 720 ggcccatgga aaaaaacttt attaaaagaagaaaaattcc cagatgtttt atggagagcc 780 agttggtctt tgtcaggtaa tgtactagctctttccggtg gcgataataa agttacttta 840 tggaaggaaa atcttgaggg taaatgggaacccgctggtg aagttcatca gtga 894 55 1273 PRT Saccharomyces cerevisiae 55Met Val Lys Leu Ala Glu Phe Ser Arg Thr Ala Thr Phe Ala Trp Ser 1 5 1015 His Asp Lys Ile Pro Leu Leu Val Ser Gly Thr Val Ser Gly Thr Val 20 2530 Asp Ala Asn Phe Ser Thr Asp Ser Ser Leu Glu Leu Trp Ser Leu Leu 35 4045 Ala Ala Asp Ser Glu Lys Pro Ile Ala Ser Leu Gln Val Asp Ser Lys 50 5560 Phe Asn Asp Leu Asp Trp Ser His Asn Asn Lys Ile Ile Ala Gly Ala 65 7075 80 Leu Asp Asn Gly Ser Leu Glu Leu Tyr Ser Thr Asn Glu Ala Asn Asn 8590 95 Ala Ile Asn Ser Met Ala Arg Phe Ser Asn His Ser Ser Ser Val Lys100 105 110 Thr Val Lys Phe Asn Ala Lys Gln Asp Asn Val Leu Ala Ser GlyGly 115 120 125 Asn Asn Gly Glu Ile Phe Ile Trp Asp Met Asn Lys Cys ThrGlu Ser 130 135 140 Pro Ser Asn Tyr Thr Pro Leu Thr Pro Gly Gln Ser MetSer Ser Val 145 150 155 160 Asp Glu Val Ile Ser Leu Ala Trp Asn Gln SerLeu Ala His Val Phe 165 170 175 Ala Ser Ala Gly Ser Ser Asn Phe Ala SerIle Trp Asp Leu Lys Ala 180 185 190 Lys Lys Glu Val Ile His Leu Ser TyrThr Ser Pro Asn Ser Gly Ile 195 200 205 Lys Gln Gln Leu Ser Val Val GluTrp His Pro Lys Asn Ser Thr Arg 210 215 220 Val Ala Thr Ala Thr Gly SerAsp Asn Asp Pro Ser Ile Leu Ile Trp 225 230 235 240 Asp Leu Arg Asn AlaAsn Thr Pro Leu Gln Thr Leu Asn Gln Gly His 245 250 255 Gln Lys Gly IleLeu Ser Leu Asp Trp Cys His Gln Asp Glu His Leu 260 265 270 Leu Leu SerSer Gly Arg Asp Asn Thr Val Leu Leu Trp Asn Pro Glu 275 280 285 Ser AlaGlu Gln Leu Ser Gln Phe Pro Ala Arg Gly Asn Trp Cys Phe 290 295 300 LysThr Lys Phe Ala Pro Glu Ala Pro Asp Leu Phe Ala Cys Ala Ser 305 310 315320 Phe Asp Asn Lys Ile Glu Val Gln Thr Leu Gln Asn Leu Thr Asn Thr 325330 335 Leu Asp Glu Gln Glu Thr Glu Thr Lys Gln Gln Glu Ser Glu Thr Asp340 345 350 Phe Trp Asn Asn Val Ser Arg Glu Glu Ser Lys Glu Lys Pro SerVal 355 360 365 Phe His Leu Gln Ala Pro Thr Trp Tyr Gly Glu Pro Ser ProAla Ala 370 375 380 His Trp Ala Phe Gly Gly Lys Leu Val Gln Ile Thr ProAsp Gly Lys 385 390 395 400 Gly Val Ser Ile Thr Asn Pro Lys Ile Ser GlyLeu Glu Ser Asn Thr 405 410 415 Thr Leu Ser Glu Ala Leu Lys Thr Lys AspPhe Lys Pro Leu Ile Asn 420 425 430 Gln Arg Leu Val Lys Val Ile Asp AspVal Asn Glu Glu Asp Trp Asn 435 440 445 Leu Leu Glu Lys Leu Ser Met AspGly Thr Glu Glu Phe Leu Lys Glu 450 455 460 Ala Leu Ala Phe Asp Asn AspGlu Ser Asp Ala Gln Asp Asp Ala Asn 465 470 475 480 Asn Glu Lys Glu AspAsp Gly Glu Glu Phe Phe Gln Gln Ile Glu Thr 485 490 495 Asn Phe Gln ProGlu Gly Asp Phe Ser Leu Ser Gly Asn Ile Glu Gln 500 505 510 Thr Ile SerLys Asn Leu Val Ser Gly Asn Ile Lys Ser Ala Val Lys 515 520 525 Asn SerLeu Glu Asn Asp Leu Leu Met Glu Ala Met Val Ile Ala Leu 530 535 540 AspSer Asn Asn Glu Arg Leu Lys Glu Ser Val Lys Asn Ala Tyr Phe 545 550 555560 Ala Lys Tyr Gly Ser Lys Ser Ser Leu Ser Arg Ile Leu Tyr Ser Ile 565570 575 Ser Lys Arg Glu Val Asp Asp Leu Val Glu Asn Leu Asp Val Ser Gln580 585 590 Trp Lys Phe Ile Ser Lys Ala Ile Gln Asn Leu Tyr Pro Asn AspIle 595 600 605 Ala Gln Arg Asn Glu Met Leu Ile Lys Leu Gly Asp Arg LeuLys Glu 610 615 620 Asn Gly His Arg Gln Asp Ser Leu Thr Leu Tyr Leu AlaAla Gly Ser 625 630 635 640 Leu Asp Lys Val Ala Ser Ile Trp Leu Ser GluPhe Pro Asp Leu Glu 645 650 655 Asp Lys Leu Lys Lys Asp Asn Lys Thr IleTyr Glu Ala His Ser Glu 660 665 670 Cys Leu Thr Glu Phe Ile Glu Arg PheThr Val Phe Ser Asn Phe Ile 675 680 685 Asn Gly Ser Ser Thr Ile Asn AsnGlu Gln Leu Ile Ala Lys Phe Leu 690 695 700 Glu Phe Ile Asn Leu Thr ThrSer Thr Gly Asn Phe Glu Leu Ala Thr 705 710 715 720 Glu Phe Leu Asn SerLeu Pro Ser Asp Asn Glu Glu Val Lys Thr Glu 725 730 735 Lys Ala Arg ValLeu Ile Ala Ser Gly Lys Ser Leu Pro Ala Gln Asn 740 745 750 Pro Ala ThrAla Thr Thr Ser Lys Ala Lys Tyr Thr Asn Ala Lys Thr 755 760 765 Asn LysAsn Val Pro Val Leu Pro Thr Pro Gly Met Pro Ser Thr Thr 770 775 780 SerIle Pro Ser Met Gln Ala Pro Phe Tyr Gly Met Thr Pro Gly Ala 785 790 795800 Ser Ala Asn Ala Leu Pro Pro Lys Pro Tyr Val Pro Ala Thr Thr Thr 805810 815 Ser Ala Pro Val His Thr Glu Gly Lys Tyr Ala Pro Pro Ser Gln Pro820 825 830 Ser Met Ala Ser Pro Phe Val Asn Lys Thr Asn Ser Ser Thr ArgLeu 835 840 845 Asn Ser Phe Ala Pro Pro Pro Asn Pro Tyr Ala Thr Ala ThrVal Pro 850 855 860 Ala Thr Asn Val Ser Thr Thr Ser Ile Pro Gln Asn ThrPhe Ala Pro 865 870 875 880 Ile Gln Pro Gly Met Pro Ile Met Gly Asp TyrAsn Ala Gln Ser Ser 885 890 895 Ser Ile Pro Ser Gln Pro Pro Ile Asn AlaVal Ser Gly Gln Thr Pro 900 905 910 His Leu Asn Arg Lys Ala Asn Asp GlyTrp Asn Asp Leu Pro Leu Lys 915 920 925 Val Lys Glu Lys Pro Ser Arg AlaLys Ala Val Ser Val Ala Pro Pro 930 935 940 Asn Ile Leu Ser Thr Pro ThrPro Leu Asn Gly Ile Pro Ala Asn Ala 945 950 955 960 Ala Ser Thr Met ProPro Pro Pro Leu Ser Arg Ala Pro Ser Ser Val 965 970 975 Ser Met Val SerPro Pro Pro Leu His Lys Asn Ser Arg Val Pro Ser 980 985 990 Leu Val AlaThr Ser Glu Ser Pro Arg Ala Ser Ile Ser Asn Pro Tyr 995 1000 1005 AlaPro Pro Gln Ser Ser Gln Gln Phe Pro Ile Gly Thr Ile Ser 1010 1015 1020Thr Ala Asn Gln Thr Ser Asn Thr Ala Gln Val Ala Ser Ser Asn 1025 10301035 Pro Tyr Ala Pro Pro Pro Gln Gln Arg Val Ala Thr Pro Leu Ser 10401045 1050 Gly Gly Val Pro Pro Ala Pro Leu Pro Lys Ala Ser Asn Pro Tyr1055 1060 1065 Ala Pro Thr Ala Thr Thr Gln Pro Asn Gly Ser Ser Tyr ProPro 1070 1075 1080 Thr Gly Pro Tyr Thr Asn Asn His Thr Met Thr Ser ProPro Pro 1085 1090 1095 Val Phe Asn Lys Pro Pro Thr Gly Pro Pro Pro IleSer Met Lys 1100 1105 1110 Lys Arg Ser Asn Lys Leu Ala Ser Ile Glu GlnAsn Pro Ser Gln 1115 1120 1125 Gly Ala Thr Tyr Pro Pro Thr Leu Ser SerSer Ala Ser Pro Leu 1130 1135 1140 Gln Pro Ser Gln Pro Pro Thr Leu AlaSer Gln Val Asn Thr Ser 1145 1150 1155 Ala Glu Asn Val Ser His Glu IlePro Ala Asp Gln Gln Pro Ile 1160 1165 1170 Val Asp Phe Leu Lys Glu GluLeu Ala Arg Val Thr Pro Leu Thr 1175 1180 1185 Pro Lys Glu Tyr Ser LysGln Leu Lys Asp Cys Asp Lys Arg Leu 1190 1195 1200 Lys Ile Leu Phe TyrHis Leu Glu Lys Gln Asp Leu Leu Thr Gln 1205 1210 1215 Pro Thr Ile AspCys Leu His Asp Leu Val Ala Leu Met Lys Glu 1220 1225 1230 Lys Lys TyrLys Glu Ala Met Val Ile His Ala Asn Ile Ala Thr 1235 1240 1245 Asn HisAla Gln Glu Gly Gly Asn Trp Leu Thr Gly Val Lys Arg 1250 1255 1260 LeuIle Gly Ile Ala Glu Ala Thr Leu Asn 1265 1270 56 2000 DNA Saccharomycescerevisiae 56 atggtcaaac ttgctgagtt ttctcgaaca gccacgtttg cgtggtcacatgataaaatt 60 ccattattgg tctctggtac cgtatctggt acggtggatg ctaatttctccactgattca 120 tctctagaat tgtggtcatt gttggctgct gattcggaga agcctattgcttccttgcaa 180 gtggattcca aattcaatga tttggattgg tctcataata acaagattattgctggtgct 240 ctggataacg gtagtttgga attgtactcc accaatgaag caaacaacgctatcaactcc 300 atggccagat ttagcaacca ttcttcctct gtgaagacgg taaagtttaacgcaaagcaa 360 gacaacgttc ttgcttcggg tggtaacaac ggtgaaattt ttatttgggacatgaataaa 420 tgcactgaat cgccctccaa ttatactcca ttgacaccgg gtcaatcgatgtcgtccgtt 480 gacgaggtca tttccctagc atggaaccaa tctttggccc atgtttttgcatctgccggg 540 tcgtctaatt tcgcatctat ttgggatttg aaggctaaga aggaagtcattcatctaagt 600 tacacttcac ctaattcagg tatcaagcaa cagctgtccg ttgttgaatggcacccaaaa 660 aactccacaa gagtggcaac ggctactggt agcgataatg atccatctatcctgatctgg 720 gatttaagaa acgccaacac accattgcag actttaaatc aaggccatcaaaagggtatt 780 ttgtcattag attggtgtca tcaggacgaa catctattat tgtccagtggtagagataat 840 accgttcttc tatggaaccc tgagtcagcc gaacaactgt cccaattcccagctcgtgga 900 aactggtgtt ttaagaccaa atttgcacca gaggctccag acctatttgcttgtgcctcc 960 tttgataaca aaattgaggt acagactttg caaaatctca caaacactttggatgagcaa 1020 gaaaccgaaa ctaagcagca agaatctgaa acagattttt ggaataatgtttcccgagag 1080 gaatcaaaag agaagccatc tgttttccat ttacaagccc caacttggtatggggaacca 1140 tctcccgcag ctcattgggc tttcggtggt aaattggttc aaattactccagatggtaaa 1200 ggtgtatcta taacaaaccc aaaaatttca ggcttagaat caaacactactttgagtgaa 1260 gcgttgaaaa ctaaggattt caaaccatta ataaatcaaa gactggtcaaagttattgat 1320 gacgttaatg aagaagattg gaatttattg gaaaagttat caatggacggtactgaggag 1380 ttcttgaaag aggctcttgc attcgacaac gatgaatcag atgcacaagacgatgccaac 1440 aatgagaaag aagacgatgg ggaagaattc tttcaacaaa ttgaaaccaatttccaaccc 1500 gagggcgatt tctccttgtc tggtaatatc gaacaaacta tttccaagaacttggtttct 1560 ggcaacatta agagcgctgt gaaaaattct ctagagaatg acttactaatggaggccatg 1620 gtgatcgcat tagattcaaa taacgaaaga ttaaaggaaa gtgtcaagaatgcctatttt 1680 gcgaagtatg gatctaaatc atcgctctcg aggatactat actccatttctaagagggaa 1740 gtagatgatt tggttgaaaa tttggatgtc tctcagtgga agtttatctctaaagcaatt 1800 caaaacttat atccaaatga tatcgcccag aggaatgaaa tgttgattaaattgggagac 1860 aggttaaagg aaaatggtca tagacaagat tctttgactt tgtacttggctgccggatca 1920 ttagataagg tggcttcaat ttggttatca gaatttccag atttggaggataaattgaag 1980 aaagataata agacaattta 2000 57 649 PRT Saccharomycescerevisiae 57 Met Ser Arg Ala Val Gly Ile Asp Leu Gly Thr Thr Tyr SerCys Val 1 5 10 15 Ala His Phe Ser Asn Asp Arg Val Glu Ile Ile Ala AsnAsp Gln Gly 20 25 30 Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp ThrGlu Arg Leu 35 40 45 Ile Gly Asp Ala Ala Lys Asn Gln Ala Ala Ile Asn ProHis Asn Thr 50 55 60 Val Phe Asp Ala Lys Arg Leu Ile Gly Arg Lys Phe AspAsp Pro Glu 65 70 75 80 Val Thr Thr Asp Ala Lys His Phe Pro Phe Lys ValIle Ser Arg Asp 85 90 95 Gly Lys Pro Val Val Gln Val Glu Tyr Lys Gly GluThr Lys Thr Phe 100 105 110 Thr Pro Glu Glu Ile Ser Ser Met Val Leu SerLys Met Lys Glu Thr 115 120 125 Ala Glu Asn Tyr Leu Gly Thr Thr Val AsnAsp Ala Val Val Thr Val 130 135 140 Pro Ala Tyr Phe Asn Asp Ser Gln ArgGln Ala Thr Lys Asp Ala Gly 145 150 155 160 Thr Ile Ala Gly Met Asn ValLeu Arg Ile Ile Asn Glu Pro Thr Ala 165 170 175 Ala Ala Ile Ala Tyr GlyLeu Asp Lys Lys Gly Arg Ala Glu His Asn 180 185 190 Val Leu Ile Phe AspLeu Gly Gly Gly Thr Phe Asp Val Ser Leu Leu 195 200 205 Ser Ile Asp GluGly Val Phe Glu Val Lys Ala Thr Ala Gly Asp Thr 210 215 220 His Leu GlyGly Glu Asp Phe Asp Asn Arg Leu Val Asn His Leu Ala 225 230 235 240 ThrGlu Phe Lys Arg Lys Thr Lys Lys Asp Ile Ser Asn Asn Gln Arg 245 250 255Ser Leu Arg Arg Leu Arg Thr Ala Ala Glu Arg Ala Lys Arg Ala Leu 260 265270 Ser Ser Ser Ser Gln Thr Ser Ile Glu Ile Asp Ser Leu Phe Glu Gly 275280 285 Met Asp Phe Tyr Thr Ser Leu Thr Arg Ala Arg Phe Glu Glu Leu Cys290 295 300 Ala Asp Leu Phe Arg Ser Thr Leu Glu Pro Val Glu Lys Val LeuLys 305 310 315 320 Asp Ser Lys Leu Asp Lys Ser Gln Ile Asp Glu Ile ValLeu Val Gly 325 330 335 Gly Ser Thr Arg Ile Pro Lys Ile Gln Lys Leu ValSer Asp Phe Phe 340 345 350 Asn Gly Lys Glu Pro Asn Arg Ser Ile Asn ProAsp Glu Ala Val Ala 355 360 365 Tyr Gly Ala Ala Val Gln Ala Ala Ile LeuThr Gly Asp Gln Ser Thr 370 375 380 Lys Thr Gln Asp Leu Leu Leu Leu AspVal Ala Pro Leu Ser Leu Gly 385 390 395 400 Ile Glu Thr Ala Gly Gly IleMet Thr Lys Leu Ile Pro Arg Asn Ser 405 410 415 Thr Ile Pro Thr Lys LysSer Glu Thr Phe Ser Thr Tyr Ala Asp Asn 420 425 430 Gln Pro Gly Val LeuIle Gln Val Phe Glu Gly Glu Arg Thr Arg Thr 435 440 445 Lys Asp Asn AsnLeu Leu Gly Lys Phe Glu Leu Ser Gly Ile Pro Pro 450 455 460 Ala Pro ArgGly Val Pro Gln Ile Asp Val Thr Phe Asp Ile Asp Ala 465 470 475 480 AsnGly Ile Leu Asn Val Ser Ala Leu Glu Lys Gly Thr Gly Lys Ser 485 490 495Asn Lys Ile Thr Ile Thr Asn Asp Lys Gly Arg Leu Ser Lys Asp Asp 500 505510 Ile Asp Arg Met Val Ser Glu Ala Glu Lys Tyr Arg Ala Asp Asp Glu 515520 525 Arg Glu Ala Glu Arg Val Gln Ala Lys Asn Gln Leu Glu Ser Tyr Ala530 535 540 Phe Thr Leu Lys Asn Thr Ile Asn Glu Ala Ser Phe Lys Glu LysVal 545 550 555 560 Gly Glu Asp Asp Ala Lys Arg Leu Glu Thr Ala Ser GlnGlu Thr Ile 565 570 575 Asp Trp Leu Asp Ala Ser Gln Ala Ala Ser Thr AspGlu Tyr Lys Asp 580 585 590 Arg Gln Lys Glu Leu Glu Gly Ile Ala Asn ProIle Met Thr Lys Phe 595 600 605 Tyr Gly Ala Gly Ala Gly Ala Gly Pro GlyAla Gly Glu Ser Gly Gly 610 615 620 Phe Pro Gly Ser Met Pro Asn Ser GlyAla Thr Gly Gly Gly Glu Asp 625 630 635 640 Thr Gly Pro Thr Val Glu GluVal Asp 645 58 1950 DNA Saccharomyces cerevisiae 58 atgtctagagcagttggtat tgatttggga acaacttact cgtgtgttgc tcatttttcc 60 aatgatagggtagagataat tgcaaatgat caaggtaata ggaccactcc atcgtatgtg 120 gctttcacagacaccgaaag attaattggt gacgccgcca aaaatcaagc tgcaatcaat 180 cctcataatacagtttttga tgcaaagcgg ttaattggtc gtaaatttga tgatcctgaa 240 gtgacgacagatgccaagca cttccctttc aaagttatat ccagagatgg taaacctgta 300 gtgcaagtagaatataaggg tgaaacgaaa acatttacgc ctgaggaaat ttcttccatg 360 gttttaagcaaaatgaagga aactgctgag aactatttgg gaactacggt caatgatgct 420 gttgtaactgttcctgcata tttcaatgat tctcaaagac aagccactaa ggatgcagga 480 actattgcagggatgaacgt tttacgtatt atcaatgaac ccactgcagc agcaattgct 540 tatggcttggataagaaagg cagggctgag cacaatgtcc tgatttttga tttgggtggt 600 ggtacttttgacgtctcttt actttcaatt gatgagggtg tttttgaggt taaggctacc 660 gcaggagacactcatttagg tggtgaagat tttgataata ggttggtgaa ccatttagcc 720 actgaattcaaaaggaaaac gaaaaaggac atctctaata atcaaagatc gttaagaaga 780 ttgagaactgcggcagaaag agctaagaga gcgctttctt cctcatctca aacctcgatc 840 gagatcgattctttatttga aggtatggat ttctacactt cgttaacaag ggcaaggttt 900 gaagagctatgtgctgattt attcagatcc acattggaac cagtagaaaa ggttcttaaa 960 gattcgaagctggacaagtc ccaaattgat gagattgtgt tagtcggtgg atctaccaga 1020 atcccaaagattcagaaatt agtttctgac ttcttcaatg gcaaagagcc taatcgttct 1080 atcaacccggatgaggctgt tgcttatggt gcagccgttc aagctgccat tttaaccggc 1140 gatcaatcaacaaagacaca agatttacta ttattggatg ttgcgccatt gtccctagga 1200 attgaaactgcaggcggcat aatgactaag ctaattccta gaaactcaac gattccaaca 1260 aagaaatcggaaaccttctc tacctatgca gataatcaac ctggtgtttt aattcaagtc 1320 tttgaaggtgaaagaacaag aacaaaggat aataacttac ttggtaaatt cgaattaagt 1380 ggcattccgcctgctcccag aggtgtgcct caaattgatg ttacctttga tatcgacgct 1440 aatggtattcttaatgtgtc tgctttggaa aagggtactg gtaagagtaa caaaatcacg 1500 atcactaacgataaaggtag gctctcgaag gatgatattg ataggatggt ttctgaagct 1560 gaaaaatatagggctgacga tgaaagggag gcagaacgag ttcaggctaa gaatcagctt 1620 gaatcgtatgcatttacttt gaagaatacc ataaacgaag caagtttcaa agagaaagta 1680 ggtgaagatgatgcaaagag attagaaaca gcgtctcagg aaaccattga ctggttagat 1740 gcatcgcaggcagcctctac ggacgaatat aaggatagac aaaaggagtt ggaaggcatt 1800 gccaatccaataatgacgaa attttacggt gctggtgccg gcgcaggtcc tggagcgggg 1860 gaatccggtggattccccgg atccatgccc aactcgggtg ctacgggagg tggagaagat 1920 acaggtccaacagtggaaga ggttgattga 1950 59 206 PRT Saccharomyces cerevisiae 59 MetPro Ser His Arg Asn Ser Asn Leu Lys Phe Cys Thr Val Cys Ala 1 5 10 15Ser Asn Asn Asn Arg Ser Met Glu Ser His Lys Val Leu Gln Glu Ala 20 25 30Gly Tyr Asn Val Ser Ser Tyr Gly Thr Gly Ser Ala Val Arg Leu Pro 35 40 45Gly Leu Ser Ile Asp Lys Pro Asn Val Tyr Ser Phe Gly Thr Pro Tyr 50 55 60Asn Asp Ile Tyr Asn Asp Leu Leu Ser Gln Ser Ala Asp Arg Tyr Lys 65 70 7580 Ser Asn Gly Leu Leu Gln Met Leu Asp Arg Asn Arg Arg Leu Lys Lys 85 9095 Ala Pro Glu Lys Trp Gln Glu Ser Thr Lys Val Phe Asp Phe Val Phe 100105 110 Thr Cys Glu Glu Arg Cys Phe Asp Ala Val Cys Glu Asp Leu Met Asn115 120 125 Arg Gly Gly Lys Leu Asn Lys Ile Val His Val Ile Asn Val AspIle 130 135 140 Lys Asp Asp Asp Glu Asn Ala Lys Ile Gly Ser Lys Ala IleLeu Glu 145 150 155 160 Leu Ala Asp Met Leu Asn Asp Lys Ile Glu Gln CysGlu Lys Asp Asp 165 170 175 Ile Pro Phe Glu Asp Cys Ile Met Asp Ile LeuThr Glu Trp Gln Ser 180 185 190 Ser His Ser Gln Leu Pro Ser Leu Tyr AlaPro Ser Tyr Tyr 195 200 205 60 621 DNA Saccharomyces cerevisiae 60atgcctagtc atcgcaattc aaacttgaag ttttgcacag tttgtgcatc aaacaacaat 60cgttcaatgg aatcgcataa agtcctgcaa gaagcaggct ataatgttag ctcttacgga 120acaggttcag ctgtgagact gcctggtcta tcgatagata agcctaatgt gtactcattt 180ggtacaccct ataatgatat atataatgat cttttatcac aatcagcaga ccgttacaag 240tcgaacggtt tattgcaaat gctggatcgt aatagaagac tcaaaaaagc acctgaaaaa 300tggcaagaaa gtacaaaagt cttcgacttc gttttcactt gtgaagagag atgttttgat 360gccgtttgtg aagatttgat gaatagaggt gggaaattaa acaaaatagt gcatgtaatt 420aatgttgaca ttaaagatga tgatgaaaat gctaaaattg gtagcaaagc tatattggaa 480ttagctgata tgctcaatga taaaatagaa caatgtgaaa aagatgacat tccctttgaa 540gattgtataa tggacatttt aactgagtgg caaagctcac attctcaact accgtcatta 600tacgctcctt catattacta a 621 61 516 PRT Saccharomyces cerevisiae 61 MetSer Thr Gln Gln Gln Ser Tyr Thr Ile Trp Ser Pro Gln Asp Thr 1 5 10 15Val Lys Asp Val Ala Glu Ser Leu Gly Leu Glu Asn Ile Asn Asp Asp 20 25 30Val Leu Lys Ala Leu Ala Met Asp Val Glu Tyr Arg Ile Leu Glu Ile 35 40 45Ile Glu Gln Ala Val Lys Phe Lys Arg His Ser Lys Arg Asp Val Leu 50 55 60Thr Thr Asp Asp Val Ser Lys Ala Leu Arg Val Leu Asn Val Glu Pro 65 70 7580 Leu Tyr Gly Tyr Tyr Asp Gly Ser Glu Val Asn Lys Ala Val Ser Phe 85 9095 Ser Lys Val Asn Thr Ser Gly Gly Gln Ser Val Tyr Tyr Leu Asp Glu 100105 110 Glu Glu Val Asp Phe Asp Arg Leu Ile Asn Glu Pro Leu Pro Gln Val115 120 125 Pro Arg Leu Pro Thr Phe Thr Thr His Trp Leu Ala Val Glu GlyVal 130 135 140 Gln Pro Ala Ile Ile Gln Asn Pro Asn Leu Asn Asp Ile ArgVal Ser 145 150 155 160 Gln Pro Pro Phe Ile Arg Gly Ala Ile Val Thr AlaLeu Asn Asp Asn 165 170 175 Ser Leu Gln Thr Pro Val Thr Ser Thr Thr AlaSer Ala Ser Val Thr 180 185 190 Asp Thr Gly Ala Ser Gln His Leu Ser AsnVal Lys Pro Gly Gln Asn 195 200 205 Thr Glu Val Lys Pro Leu Val Lys HisVal Leu Ser Lys Glu Leu Gln 210 215 220 Ile Tyr Phe Asn Lys Val Ile SerThr Leu Thr Ala Lys Ser Gln Ala 225 230 235 240 Asp Glu Ala Ala Gln HisMet Lys Gln Ala Ala Leu Thr Ser Leu Arg 245 250 255 Thr Asp Ser Gly LeuHis Gln Leu Val Pro Tyr Phe Ile Gln Phe Ile 260 265 270 Ala Glu Gln IleThr Gln Asn Leu Ser Asp Leu Gln Leu Leu Thr Thr 275 280 285 Ile Leu GluMet Ile Tyr Ser Leu Leu Ser Asn Thr Ser Ile Phe Leu 290 295 300 Asp ProTyr Ile His Ser Leu Met Pro Ser Ile Leu Thr Leu Leu Leu 305 310 315 320Ala Lys Lys Leu Gly Gly Ser Pro Lys Asp Asp Ser Pro Gln Glu Ile 325 330335 His Glu Phe Leu Glu Arg Thr Asn Ala Leu Arg Asp Phe Ala Ala Ser 340345 350 Leu Leu Asp Tyr Val Leu Lys Lys Phe Pro Gln Ala Tyr Lys Ser Leu355 360 365 Lys Pro Arg Val Thr Arg Thr Leu Leu Lys Thr Phe Leu Asp IleAsn 370 375 380 Arg Val Phe Gly Thr Tyr Tyr Gly Cys Leu Lys Gly Val SerVal Leu 385 390 395 400 Glu Gly Glu Ser Ile Arg Phe Phe Leu Gly Asn LeuAsn Asn Trp Ala 405 410 415 Arg Leu Val Phe Asn Glu Ser Gly Ile Thr LeuAsp Asn Ile Glu Glu 420 425 430 His Leu Asn Asp Asp Ser Asn Pro Thr ArgThr Lys Phe Thr Lys Glu 435 440 445 Glu Thr Gln Ile Leu Val Asp Thr ValIle Ser Ala Leu Leu Val Leu 450 455 460 Lys Lys Asp Leu Pro Asp Leu TyrGlu Gly Lys Gly Glu Lys Val Thr 465 470 475 480 Asp Glu Asp Lys Glu LysLeu Leu Glu Arg Cys Gly Val Thr Ile Gly 485 490 495 Phe His Ile Leu LysArg Asp Asp Ala Lys Glu Leu Ile Ser Ala Ile 500 505 510 Phe Phe Gly Glu515 62 1551 DNA Saccharomyces cerevisiae 62 atgtctacac agcagcaatcttacacaatt tggtctccac aagatactgt taaagatgtc 60 gctgagtcgc ttgggttggaaaatatcaac gatgacgtgt tgaaagcact tgctatggac 120 gttgaatacc gtattctagagattattgaa caggcagtca agtttaaaag acactctaaa 180 agagatgttt taactacagacgatgtatcg aaggccctgc gtgttctgaa tgtggaacct 240 ttatatggat attatgatggctctgaagtt aataaagctg tatcatttag taaggttaac 300 acaagtggag gacagtcggtttactacctg gatgaggaag aagtagattt tgatagatta 360 ataaatgagc cattaccgcaagtgcctcgt ctaccaactt ttactacaca ttggctagca 420 gttgaagggg ttcaacctgccataatccaa aatccaaatt tgaatgatat aagggtatcc 480 caaccaccat ttattagaggtgctattgtc actgctttga atgataacag cctccaaaca 540 cctgtaacgt cgacgacagcaagtgcttct gtgacggata caggcgcctc tcaacacctg 600 tctaatgtta aaccaggacagaatactgaa gtcaaacctt tagtgaaaca cgttttatcc 660 aaagaattac agatttatttcaataaggtc atttcaacct tgacagcgaa gagtcaagct 720 gatgaagctg cacagcatatgaaacaagca gctttgactt cactacgaac tgatagtggc 780 ctgcaccaac tggttccatattttattcaa tttattgccg aacaaatcac acaaaatctt 840 tccgatttac aattgctaacaacaattttg gaaatgattt actctttact aagcaatact 900 tctattttcc tggacccttatattcattcg ttaatgcctt ccattttaac cttgctatta 960 gcaaaaaaac ttggtggatcaccaaaagat gattcgccgc aagaaattca tgaattttta 1020 gaaagaacga atgcactacgtgattttgcc gcatctttgt tggactatgt attgaaaaaa 1080 tttcctcaag catacaaatccctgaagcca agagtaacta ggacactgct aaagacattt 1140 ttggacataa accgtgtttttgggacttac tatgggtgct taaagggtgt atcggtacta 1200 gaaggtgaat ccatcagattcttcttagga aacctaaata attgggcgcg cttggtattc 1260 aatgaaagcg gaataactctagacaatata gaggaacatt tgaacgatga ctccaatcca 1320 actaggacca aatttactaaagaagaaact caaatcttgg ttgatactgt aattagtgca 1380 ttgttagttc taaaaaaggatttaccagat ttgtacgagg gtaaaggtga aaaagttacg 1440 gatgaggata aggaaaaattgctagagagg tgtggtgtca ctattggatt tcacattttg 1500 aaaagagacg acgcaaaagaattaattagt gcgatatttt ttggcgaata g 1551 63 914 PRT Saccharomycescerevisiae 63 Met Thr Asp Gln Arg Gly Pro Pro Pro Pro His Pro Gln GlnAla Asn 1 5 10 15 Gly Tyr Lys Lys Phe Pro Pro His Asp Asn Gln Tyr SerGly Ala Asn 20 25 30 Asn Ser Gln Pro Asn Asn His Tyr Asn Glu Asn Leu TyrSer Ala Arg 35 40 45 Glu Pro His Asn Asn Lys Gln Tyr Gln Ser Lys Asn GlyLys Tyr Gly 50 55 60 Thr Asn Lys Tyr Asn Asn Arg Asn Asn Ser Gln Gly AsnAla Gln Tyr 65 70 75 80 Tyr Asn Asn Arg Phe Asn Asn Gly Tyr Arg Leu AsnAsn Asn Asp Tyr 85 90 95 Asn Pro Ala Met Leu Pro Gly Met Gln Trp Pro AlaAsn Tyr Tyr Ala 100 105 110 Pro Gln Met Tyr Tyr Ile Pro Gln Gln Met ValPro Val Ala Ser Pro 115 120 125 Pro Tyr Thr His Gln Pro Leu Asn Thr AsnPro Glu Pro Pro Ser Thr 130 135 140 Pro Lys Thr Thr Lys Ile Glu Ile ThrThr Lys Thr Gly Glu Arg Leu 145 150 155 160 Asn Leu Lys Lys Phe His GluGlu Lys Lys Ala Ser Lys Gly Glu Glu 165 170 175 Lys Asn Asp Gly Val GluGln Lys Ser Lys Ser Gly Thr Pro Phe Glu 180 185 190 Lys Glu Ala Thr ProVal Leu Pro Ala Asn Glu Ala Val Lys Asp Thr 195 200 205 Leu Thr Glu ThrSer Asn Glu Lys Ser Thr Ser Glu Ala Glu Asn Thr 210 215 220 Lys Arg LeuPhe Leu Glu Gln Val Arg Leu Arg Lys Ala Ala Met Glu 225 230 235 240 ArgLys Lys Asn Gly Leu Ile Ser Glu Thr Glu Lys Lys Gln Glu Thr 245 250 255Ser Asn His Asp Asn Thr Asp Thr Thr Lys Pro Asn Ser Val Ile Glu 260 265270 Ser Glu Pro Ile Lys Glu Ala Pro Lys Pro Thr Gly Glu Ala Asn Glu 275280 285 Val Val Ile Asp Gly Lys Ser Gly Ala Ser Val Lys Thr Pro Gln His290 295 300 Val Thr Gly Ser Val Thr Lys Ser Val Thr Phe Asn Glu Pro GluAsn 305 310 315 320 Glu Ser Ser Ser Gln Asp Val Asp Glu Leu Val Lys AspAsp Asp Thr 325 330 335 Thr Glu Ile Ser Asp Thr Thr Gly Gly Lys Thr ValAsn Lys Ser Asp 340 345 350 Asp Glu Thr Ile Asn Ser Val Ile Thr Thr GluGlu Asn Thr Val Lys 355 360 365 Glu Thr Glu Pro Ser Thr Ser Asp Ile GluMet Pro Thr Val Ser Gln 370 375 380 Leu Leu Glu Thr Leu Gly Lys Ala GlnPro Ile Ser Asp Ile Tyr Glu 385 390 395 400 Phe Ala Tyr Pro Glu Asn ValGlu Arg Pro Asp Ile Lys Tyr Lys Lys 405 410 415 Pro Ser Val Lys Tyr ThrTyr Gly Pro Thr Phe Leu Leu Gln Phe Lys 420 425 430 Asp Lys Leu Lys PheArg Pro Asp Pro Ala Trp Val Glu Ala Val Ser 435 440 445 Ser Lys Ile ValIle Pro Pro His Ile Ala Arg Asn Lys Pro Lys Asp 450 455 460 Ser Gly ArgPhe Gly Gly Asp Phe Arg Ser Pro Ser Met Arg Gly Met 465 470 475 480 AspHis Thr Ser Ser Ser Arg Val Ser Ser Lys Arg Arg Ser Lys Arg 485 490 495Met Gly Asp Asp Arg Arg Ser Asn Arg Gly Tyr Thr Ser Arg Lys Asp 500 505510 Arg Glu Lys Ala Ala Glu Lys Ala Glu Glu Gln Ala Pro Lys Glu Glu 515520 525 Ile Ala Pro Leu Val Pro Ser Ala Asn Arg Trp Ile Pro Lys Ser Arg530 535 540 Val Lys Lys Thr Glu Lys Lys Leu Ala Pro Asp Gly Lys Thr GluLeu 545 550 555 560 Phe Asp Lys Glu Glu Val Glu Arg Lys Met Lys Ser LeuLeu Asn Lys 565 570 575 Leu Thr Leu Glu Met Phe Asp Ser Ile Ser Ser GluIle Leu Asp Ile 580 585 590 Ala Asn Gln Ser Lys Trp Glu Asp Asp Gly GluThr Leu Lys Ile Val 595 600 605 Ile Glu Gln Ile Phe His Lys Ala Cys AspGlu Pro His Trp Ser Ser 610 615 620 Met Tyr Ala Gln Leu Cys Gly Lys ValVal Lys Asp Leu Asp Pro Asn 625 630 635 640 Ile Lys Asp Lys Glu Asn GluGly Lys Asn Gly Pro Lys Leu Val Leu 645 650 655 His Tyr Leu Val Ala ArgCys His Glu Glu Phe Glu Lys Gly Trp Ala 660 665 670 Asp Lys Leu Pro AlaGly Glu Asp Gly Asn Pro Leu Glu Pro Glu Met 675 680 685 Met Ser Asp GluTyr Tyr Ile Ala Ala Ala Ala Lys Arg Arg Gly Leu 690 695 700 Gly Leu ValArg Phe Ile Gly Tyr Leu Tyr Cys Leu Asn Leu Leu Thr 705 710 715 720 GlyLys Met Met Phe Glu Cys Phe Arg Arg Leu Met Lys Asp Leu Asn 725 730 735Asn Asp Pro Ser Glu Glu Thr Leu Glu Ser Val Ile Glu Leu Leu Asn 740 745750 Thr Val Gly Glu Gln Phe Glu His Asp Lys Phe Val Thr Pro Gln Ala 755760 765 Thr Leu Glu Gly Ser Val Leu Leu Asp Asn Leu Phe Met Leu Leu Gln770 775 780 His Ile Ile Asp Gly Gly Thr Ile Ser Asn Arg Ile Lys Phe LysLeu 785 790 795 800 Ile Asp Val Lys Glu Leu Arg Glu Ile Lys His Trp AsnSer Ala Lys 805 810 815 Lys Asp Ala Gly Pro Lys Thr Ile Gln Gln Ile HisGln Glu Glu Glu 820 825 830 Gln Leu Arg Gln Lys Lys Asn Ser Gln Arg SerAsn Ser Arg Phe Asn 835 840 845 Asn His Asn Gln Ser Asn Ser Asn Arg TyrSer Ser Asn Arg Arg Asn 850 855 860 Met Gln Asn Thr Gln Arg Asp Ser PheAla Ser Thr Lys Thr Gly Ser 865 870 875 880 Phe Arg Asn Asn Gln Arg AsnAla Arg Lys Val Glu Glu Val Ser Gln 885 890 895 Ala Pro Arg Ala Asn MetPhe Asp Ala Leu Met Asn Asn Asp Gly Asp 900 905 910 Ser Asp 64 2000 DNASaccharomyces cerevisiae 64 atgactgacc aaagaggtcc accgccccca cacccgcagcaagccaatgg ctacaagaaa 60 tttcctcctc atgataacca atactctgga gccaataatagtcagccaaa taaccactac 120 aatgaaaatc tttacagtgc aagggaacct cacaataacaagcaatacca gtcgaaaaat 180 ggcaaatacg ggacaaataa atacaataat cgtaataatagtcaaggaaa tgcacagtac 240 tacaataaca gattcaataa tggctacaga ctgaataacaacgactataa cccggcaatg 300 ttgccaggta tgcaatggcc agctaactac tatgctcctcagatgtatta tataccccaa 360 caaatggttc cagttgcttc tccaccatat acacaccaaccactcaacac taatcccgaa 420 cctccctcta cacctaaaac tacaaaaata gaaatcactacgaaaactgg tgaacgttta 480 aatttgaaaa aattccacga agaaaagaaa gcttcgaaaggtgaagagaa gaatgatggg 540 gtagagcaga aatctaaatc aggaacccct tttgaaaaggaggcaactcc tgtactacct 600 gctaatgaag cagttaaaga tacgttaacc gaaacatctaatgaaaaatc tacctctgaa 660 gcggaaaata ctaagagatt atttttagag caggtgagattgcgcaaagc tgccatggaa 720 agaaagaaaa acggtttaat ctccgaaact gagaagaagcaagaaacttc aaatcatgat 780 aatactgata caactaagcc caactcggtt attgaatctgaacctattaa ggaagcgccg 840 aaaccaacgg gagaagctaa tgaagttgtg attgatggaaaatcaggggc aagtgtcaaa 900 actccacaac atgtaactgg aagtgtaact aaatccgtgacttttaacga acccgagaat 960 gagagcagtt ctcaggatgt tgatgagctt gttaaagatgatgatactac tgaaatttcg 1020 gatacgactg gtggaaaaac tgtaaataaa agtgacgacgaaacaataaa ttccgtaatc 1080 accacagagg agaacacagt gaaggagaca gaaccttccacttccgatat tgagatgcca 1140 actgtatctc aactacttga aactttaggt aaggctcaaccaatttctga catatatgag 1200 tttgcctatc cggaaaatgt tgaaaggcct gatatcaaatacaagaaacc aagcgttaag 1260 tacacttatg gccctacctt cttactgcaa ttcaaagataaactaaaatt caggcctgat 1320 cctgcgtggg ttgaagctgt atcctcgaaa attgttatacctcctcatat agccagaaat 1380 aaaccaaaag atagtggcag atttggaggc gatttcagaagtccatctat gcgcggtatg 1440 gaccatactt ccagttcaag agtttcatcg aaaagaagatcaaagagaat gggtgacgac 1500 agaagatcta atagaggata tacctccaga aaagatcgcgaaaaagctgc agaaaaggca 1560 gaagagcaag ctccaaagga agaaatcgcc ccgttggttccgagtgctaa tagatggata 1620 cctaaatcaa gggttaaaaa aacagaaaag aagttagctcctgacggcaa aactgaatta 1680 tttgacaagg aagaagtaga acgaaaaatg aagtctctattaaacaagtt aactttagaa 1740 atgtttgatt caatctcctc tgagattttg gatatagcaaaccaatcaaa gtgggaagat 1800 gatggcgaaa cattgaaaat agttattgaa caaattttccacaaggcttg tgatgaacct 1860 cattggtctt caatgtatgc tcagttatgt ggcaaagtggtcaaagacct tgacccaaac 1920 attaaagata aagagaacga aggaaagaat ggaccaaagcttgtgttgca ttatttagtg 1980 gcaagatgtc acgaagaatt 2000 65 680 PRTSaccharomyces cerevisiae 65 Met Thr Gln Phe Thr Asp Ile Asp Lys Leu AlaVal Ser Thr Ile Arg 1 5 10 15 Ile Leu Ala Val Asp Thr Val Ser Lys AlaAsn Ser Gly His Pro Gly 20 25 30 Ala Pro Leu Gly Met Ala Pro Ala Ala HisVal Leu Trp Ser Gln Met 35 40 45 Arg Met Asn Pro Thr Asn Pro Asp Trp IleAsn Arg Asp Arg Phe Val 50 55 60 Leu Ser Asn Gly His Ala Val Ala Leu LeuTyr Ser Met Leu His Leu 65 70 75 80 Thr Gly Tyr Asp Leu Ser Ile Glu AspLeu Lys Gln Phe Arg Gln Leu 85 90 95 Gly Ser Arg Thr Pro Gly His Pro GluPhe Glu Leu Pro Gly Val Glu 100 105 110 Val Thr Thr Gly Pro Leu Gly GlnGly Ile Ser Asn Ala Val Gly Met 115 120 125 Ala Met Ala Gln Ala Asn LeuAla Ala Thr Tyr Asn Lys Pro Gly Phe 130 135 140 Thr Leu Ser Asp Asn TyrThr Tyr Val Phe Leu Gly Asp Gly Cys Leu 145 150 155 160 Gln Glu Gly IleSer Ser Glu Ala Ser Ser Leu Ala Gly His Leu Lys 165 170 175 Leu Gly AsnLeu Ile Ala Ile Tyr Asp Asp Asn Lys Ile Thr Ile Asp 180 185 190 Gly AlaThr Ser Ile Ser Phe Asp Glu Asp Val Ala Lys Arg Tyr Glu 195 200 205 AlaTyr Gly Trp Glu Val Leu Tyr Val Glu Asn Gly Asn Glu Asp Leu 210 215 220Ala Gly Ile Ala Lys Ala Ile Ala Gln Ala Lys Leu Ser Lys Asp Lys 225 230235 240 Pro Thr Leu Ile Lys Met Thr Thr Thr Ile Gly Tyr Gly Ser Leu His245 250 255 Ala Gly Ser His Ser Val His Gly Ala Pro Leu Lys Ala Asp AspVal 260 265 270 Lys Gln Leu Lys Ser Lys Phe Gly Phe Asn Pro Asp Lys SerPhe Val 275 280 285 Val Pro Gln Glu Val Tyr Asp His Tyr Gln Lys Thr IleLeu Lys Pro 290 295 300 Gly Val Glu Ala Asn Asn Lys Trp Asn Lys Leu PheSer Glu Tyr Gln 305 310 315 320 Lys Lys Phe Pro Glu Leu Gly Ala Glu LeuAla Arg Arg Leu Ser Gly 325 330 335 Gln Leu Pro Ala Asn Trp Glu Ser LysLeu Pro Thr Tyr Thr Ala Lys 340 345 350 Asp Ser Ala Val Ala Thr Arg LysLeu Ser Glu Thr Val Leu Glu Asp 355 360 365 Val Tyr Asn Gln Leu Pro GluLeu Ile Gly Gly Ser Ala Asp Leu Thr 370 375 380 Pro Ser Asn Leu Thr ArgTrp Lys Glu Ala Leu Asp Phe Gln Pro Pro 385 390 395 400 Ser Ser Gly SerGly Asn Tyr Ser Gly Arg Tyr Ile Arg Tyr Gly Ile 405 410 415 Arg Glu HisAla Met Gly Ala Ile Met Asn Gly Ile Ser Ala Phe Gly 420 425 430 Ala AsnTyr Lys Pro Tyr Gly Gly Thr Phe Leu Asn Phe Val Ser Tyr 435 440 445 AlaAla Gly Ala Val Arg Leu Ser Ala Leu Ser Gly His Pro Val Ile 450 455 460Trp Val Ala Thr His Asp Ser Ile Gly Val Gly Glu Asp Gly Pro Thr 465 470475 480 His Gln Pro Ile Glu Thr Leu Ala His Phe Arg Ser Leu Pro Asn Ile485 490 495 Gln Val Trp Arg Pro Ala Asp Gly Asn Glu Val Ser Ala Ala TyrLys 500 505 510 Asn Ser Leu Glu Ser Lys His Thr Pro Ser Ile Ile Ala LeuSer Arg 515 520 525 Gln Asn Leu Pro Gln Leu Glu Gly Ser Ser Ile Glu SerAla Ser Lys 530 535 540 Gly Gly Tyr Val Leu Gln Asp Val Ala Asn Pro AspIle Ile Leu Val 545 550 555 560 Ala Thr Gly Ser Glu Val Ser Leu Ser ValGlu Ala Ala Lys Thr Leu 565 570 575 Ala Ala Lys Asn Ile Lys Ala Arg ValVal Ser Leu Pro Asp Phe Phe 580 585 590 Thr Phe Asp Lys Gln Pro Leu GluTyr Arg Leu Ser Val Leu Pro Asp 595 600 605 Asn Val Pro Ile Met Ser ValGlu Val Leu Ala Thr Thr Cys Trp Gly 610 615 620 Lys Tyr Ala His Gln SerPhe Gly Ile Asp Arg Phe Gly Ala Ser Gly 625 630 635 640 Lys Ala Pro GluVal Phe Lys Phe Phe Gly Phe Thr Pro Glu Gly Val 645 650 655 Ala Glu ArgAla Gln Lys Thr Ile Ala Phe Tyr Lys Gly Asp Lys Leu 660 665 670 Ile SerPro Leu Lys Lys Ala Phe 675 680 66 2000 DNA Saccharomyces cerevisiae 66atgactcaat tcactgacat tgataagcta gccgtctcca ccataagaat tttggctgtg 60gacaccgtat ccaaggccaa ctcaggtcac ccaggtgctc cattgggtat ggcaccagct 120gcacacgttc tatggagtca aatgcgcatg aacccaacca acccagactg gatcaacaga 180gatagatttg tcttgtctaa cggtcacgcg gtcgctttgt tgtattctat gctacatttg 240actggttacg atctgtctat tgaagacttg aaacagttca gacagttggg ttccagaaca 300ccaggtcatc ctgaatttga gttgccaggt gttgaagtta ctaccggtcc attaggtcaa 360ggtatctcca acgctgttgg tatggccatg gctcaagcta acctggctgc cacttacaac 420aagccgggct ttaccttgtc tgacaactac acctatgttt tcttgggtga cggttgtttg 480caagaaggta tttcttcaga agcttcctcc ttggctggtc atttgaaatt gggtaacttg 540attgccatct acgatgacaa caagatcact atcgatggtg ctaccagtat ctcattcgat 600gaagatgttg ctaagagata cgaagcctac ggttgggaag ttttgtacgt agaaaatggt 660aacgaagatc tagccggtat tgccaaggct attgctcaag ctaagttatc caaggacaaa 720ccaactttga tcaaaatgac cacaaccatt ggttacggtt ccttgcatgc cggctctcac 780tctgtgcacg gtgccccatt gaaagcagat gatgttaaac aactaaagag caaattcggt 840ttcaacccag acaagtcctt tgttgttcca caagaagttt acgaccacta ccaaaagaca 900attttaaagc caggtgtcga agccaacaac aagtggaaca agttgttcag cgaataccaa 960aagaaattcc cagaattagg tgctgaattg gctagaagat tgagcggcca actacccgca 1020aattgggaat ctaagttgcc aacttacacc gccaaggact ctgccgtggc cactagaaaa 1080ttatcagaaa ctgttcttga ggatgtttac aatcaattgc cagagttgat tggtggttct 1140gccgatttaa caccttctaa cttgaccaga tggaaggaag cccttgactt ccaacctcct 1200tcttccggtt caggtaacta ctctggtaga tacattaggt acggtattag agaacacgct 1260atgggtgcca taatgaacgg tatttcagct ttcggtgcca actacaaacc atacggtggt 1320actttcttga acttcgtttc ttatgctgct ggtgccgtta gattgtccgc tttgtctggc 1380cacccagtta tttgggttgc tacacatgac tctatcggtg tcggtgaaga tggtccaaca 1440catcaaccta ttgaaacttt agcacacttc agatccctac caaacattca agtttggaga 1500ccagctgatg gtaacgaagt ttctgccgcc tacaagaact ctttagaatc caagcatact 1560ccaagtatca ttgctttgtc cagacaaaac ttgccacaat tggaaggtag ctctattgaa 1620agcgcttcta agggtggtta cgtactacaa gatgttgcta acccagatat tattttagtg 1680gctactggtt ccgaagtgtc tttgagtgtt gaagctgcta agactttggc cgcaaagaac 1740atcaaggctc gtgttgtttc tctaccagat ttcttcactt ttgacaaaca acccctagaa 1800tacagactat cagtcttacc agacaacgtt ccaatcatgt ctgttgaagt tttggctacc 1860acatgttggg gcaaatacgc tcatcaatcc ttcggtattg acagatttgg tgcctccggt 1920aaggcaccag aagtcttcaa gttcttcggt ttcaccccag aaggtgttgc tgaaagagct 1980caaaagacca ttgcattcta 2000 67 196 PRT Saccharomyces cerevisiae 67 MetVal Ala Gln Val Gln Lys Gln Ala Pro Thr Phe Lys Lys Thr Ala 1 5 10 15Val Val Asp Gly Val Phe Asp Glu Val Ser Leu Asp Lys Tyr Lys Gly 20 25 30Lys Tyr Val Val Leu Ala Phe Ile Pro Leu Ala Phe Thr Phe Val Cys 35 40 45Pro Thr Glu Ile Ile Ala Phe Ser Glu Ala Ala Lys Lys Phe Glu Glu 50 55 60Gln Gly Ala Gln Val Leu Phe Ala Ser Thr Asp Ser Glu Tyr Ser Leu 65 70 7580 Leu Ala Trp Thr Asn Ile Pro Arg Lys Glu Gly Gly Leu Gly Pro Ile 85 9095 Asn Ile Pro Leu Leu Ala Asp Thr Asn His Ser Leu Ser Arg Asp Tyr 100105 110 Gly Val Leu Ile Glu Glu Glu Gly Val Ala Leu Arg Gly Leu Phe Ile115 120 125 Ile Asp Pro Lys Gly Val Ile Arg His Ile Thr Ile Asn Asp LeuPro 130 135 140 Val Gly Arg Asn Val Asp Glu Ala Leu Arg Leu Val Glu AlaPhe Gln 145 150 155 160 Trp Thr Asp Lys Asn Gly Thr Val Leu Pro Cys AsnTrp Thr Pro Gly 165 170 175 Ala Ala Thr Ile Lys Pro Thr Val Glu Asp SerLys Glu Tyr Phe Glu 180 185 190 Ala Ala Asn Lys 195 68 591 DNASaccharomyces cerevisiae 68 atggtcgctc aagttcaaaa gcaagctcca acttttaagaaaactgccgt cgtcgacggt 60 gtctttgacg aagtctcctt ggacaaatac aagggtaagtacgttgtcct agcctttatt 120 ccattggcct tcactttcgt ctgtccaacc gaaatcattgctttctcaga agctgctaag 180 aaattcgaag aacaaggcgc tcaagttctt ttcgcctccactgactccga atactccctt 240 ttggcatgga ccaatatccc aagaaaggaa ggtggtttgggcccaatcaa cattccattg 300 ttggctgaca ccaaccactc tttgtccaga gactatggtgtcttgatcga agaagaaggt 360 gtcgccttga gaggtttgtt catcatcgac ccaaagggtgtcattagaca catcaccatt 420 aacgatttgc cagtcggtag aaacgttgac gaagccttgagattggttga agccttccaa 480 tggaccgaca agaacggtac tgtcttgcca tgtaactggactccaggtgc tgctaccatc 540 aagccaaccg ttgaagactc caaggaatac ttcgaagctgccaacaaata a 591 69 291 PRT Saccharomyces cerevisiae 69 Met Asn Ser IleLeu Asp Arg Asn Val Arg Ser Ser Glu Thr Thr Leu 1 5 10 15 Ile Lys ProGlu Ser Glu Phe Asp Asn Trp Leu Ser Asp Glu Asn Asp 20 25 30 Gly Ala SerHis Ile Asn Val Asn Lys Asp Ser Ser Ser Val Leu Ser 35 40 45 Ala Ser SerSer Thr Trp Phe Glu Pro Leu Glu Asn Ile Ile Ser Ser 50 55 60 Ala Ser SerSer Ser Ile Gly Ser Pro Ile Glu Asp Gln Phe Ile Ser 65 70 75 80 Ser AsnAsn Glu Glu Ser Ala Leu Phe Pro Thr Asp Gln Phe Phe Ser 85 90 95 Asn ProSer Ser Tyr Ser His Ser Pro Glu Val Ser Ser Ser Ile Lys 100 105 110 ArgGlu Glu Asp Asp Asn Ala Leu Ser Leu Ala Asp Phe Glu Pro Ala 115 120 125Ser Leu Gln Leu Met Pro Asn Met Ile Asn Thr Asp Asn Asn Asp Asp 130 135140 Ser Thr Pro Leu Lys Asn Glu Ile Glu Leu Asn Asp Ser Phe Ile Lys 145150 155 160 Thr Asn Leu Asp Ala Lys Glu Thr Lys Lys Arg Ala Pro Arg LysArg 165 170 175 Leu Thr Pro Phe Gln Lys Gln Ala His Asn Lys Ile Glu LysArg Tyr 180 185 190 Arg Ile Asn Ile Asn Thr Lys Ile Ala Arg Leu Gln GlnIle Ile Pro 195 200 205 Trp Val Ala Ser Glu Gln Thr Ala Phe Glu Val GlyAsp Ser Val Lys 210 215 220 Lys Gln Asp Glu Asp Gly Ala Glu Thr Ala AlaThr Thr Pro Leu Pro 225 230 235 240 Ser Ala Ala Ala Thr Ser Thr Lys LeuAsn Lys Ser Met Ile Leu Glu 245 250 255 Lys Ala Val Asp Tyr Ile Leu TyrLeu Gln Asn Asn Glu Arg Leu Tyr 260 265 270 Glu Met Glu Val Gln Arg LeuLys Ser Glu Ile Asp Thr Leu Lys Gln 275 280 285 Asp Gln Lys 290 70 876DNA Saccharomyces cerevisiae 70 atgaactcta ttttagacag aaatgttagatctagcgaaa ctactttaat taaacctgaa 60 tctgaatttg ataattggtt gtcggatgaaaatgacggag ctagtcatat caacgtcaac 120 aaggactcct cgtcagttct ttctgcatcttcttccacat ggttcgaacc attggaaaac 180 attatctcct ctgcatccag ctcctcgataggctctccaa tcgaagacca gtttatatct 240 tccaacaacg aggaatctgc tctttttccaacagatcagt ttttcagtaa tccttcctca 300 tactcgcatt ctcccgaggt tagcagctcgataaaaagag aagaggatga caatgccctt 360 tcgttggcag attttgaacc ggcttctttgcaattaatgc ctaacatgat aaatactgat 420 aataatgacg atagtacccc acttaagaatgaaatcgagc taaacgactc gtttataaaa 480 acaaatctag atgctaagga aacgaaaaagagggctccaa gaaaaagact gacccccttc 540 caaaagcaag ctcacaacaa gattgaaaaacgctacagaa taaacatcaa cacaaagatt 600 gcaagactgc agcagattat cccatgggtagcaagtgaac aaacagcttt cgaagtaggt 660 gattctgtaa aaaaacagga cgaagacggcgcagaaactg ccgctactac tcctcttcca 720 tctgccgctg ctacaagcac gaagctaaataaaagcatga tcctagaaaa agctgttgac 780 tatattctat atctacaaaa taacgaacgactatacgaaa tggaagttca aaggttgaaa 840 agtgaaatcg acactttgaa acaagaccaaaaataa 876 71 362 PRT Saccharomyces cerevisiae 71 Met Ile Asn Asn ProLys Val Asp Ser Val Ala Glu Lys Pro Lys Ala 1 5 10 15 Val Thr Ser LysGln Ser Glu Gln Ala Ala Ser Pro Glu Pro Thr Pro 20 25 30 Ala Pro Pro ValSer Arg Asn Gln Tyr Pro Ile Thr Phe Asn Leu Thr 35 40 45 Ser Thr Ala ProPhe His Leu His Asp Arg His Arg Tyr Leu Gln Glu 50 55 60 Gln Asp Leu TyrLys Cys Ala Ser Arg Asp Ser Leu Ser Ser Leu Gln 65 70 75 80 Gln Leu AlaHis Thr Pro Asn Gly Ser Thr Arg Lys Lys Tyr Ile Val 85 90 95 Glu Asp GlnSer Pro Tyr Ser Ser Glu Asn Pro Val Ile Val Thr Ser 100 105 110 Ser TyrAsn His Thr Val Cys Thr Asn Tyr Leu Arg Pro Arg Met Gln 115 120 125 PheThr Gly Tyr Gln Ile Ser Gly Tyr Lys Arg Tyr Gln Val Thr Val 130 135 140Asn Leu Lys Thr Val Asp Leu Pro Lys Lys Asp Cys Thr Ser Leu Ser 145 150155 160 Pro His Leu Ser Gly Phe Leu Ser Ile Arg Gly Leu Thr Asn Gln His165 170 175 Pro Glu Ile Ser Thr Tyr Phe Glu Ala Tyr Ala Val Asn His LysGlu 180 185 190 Leu Gly Phe Leu Ser Ser Ser Trp Lys Asp Glu Pro Val LeuAsn Glu 195 200 205 Phe Lys Ala Thr Asp Gln Thr Asp Leu Glu His Trp IleAsn Phe Pro 210 215 220 Ser Phe Arg Gln Leu Phe Leu Met Ser Gln Lys AsnGly Leu Asn Ser 225 230 235 240 Thr Asp Asp Asn Gly Thr Thr Asn Ala AlaLys Lys Leu Pro Pro Gln 245 250 255 Gln Leu Pro Thr Thr Pro Ser Ala AspAla Gly Asn Ile Ser Arg Ile 260 265 270 Phe Ser Gln Glu Lys Gln Phe AspAsn Tyr Leu Asn Glu Arg Phe Ile 275 280 285 Phe Met Lys Trp Lys Glu LysPhe Leu Val Pro Asp Ala Leu Leu Met 290 295 300 Glu Gly Val Asp Gly AlaSer Tyr Asp Gly Phe Tyr Tyr Ile Val His 305 310 315 320 Asp Gln Val ThrGly Asn Ile Gln Gly Phe Tyr Tyr His Gln Asp Ala 325 330 335 Glu Lys PheGln Gln Leu Glu Leu Val Pro Ser Leu Lys Asn Lys Val 340 345 350 Glu SerSer Asp Cys Ser Phe Glu Phe Ala 355 360 72 1089 DNA Saccharomycescerevisiae 72 atgatcaata atcctaaggt agacagtgta gcggagaaac ccaaagctgtgacatcaaag 60 cagtcggagc aagcggcttc gccagaacca acaccagccc cccctgtttctagaaatcag 120 tatccgatca cgttcaactt gacttcaacc gcaccctttc atcttcatgaccgtcatcgc 180 tacttacaag agcaagatct ttacaagtgc gcttctaggg attcgttgtcttccctgcag 240 caactcgccc atacacctaa tgggtcgaca aggaagaaat atattgttgaggaccaatct 300 ccctatagtt cggaaaatcc agtcattgtg acctcttcct ataaccatacggtttgcaca 360 aactacttaa gaccaagaat gcagtttaca gggtaccaga tatcaggatacaaacgctat 420 caggtaacag ttaacttaaa gactgtagat ttgccaaaga aagattgcacgtcgctgtcc 480 cctcatttat ctggattttt gtcgataaga ggactcacga accaacacccggaaatcagt 540 acatattttg aagcctacgc ggtaaaccac aaggaattag ggttcttgtcctctagctgg 600 aaagatgaac ctgttttaaa cgaattcaaa gccacagacc aaacagacttagaacactgg 660 ataaatttcc cctcctttag acagcttttc ctgatgagcc aaaaaaacggtctcaactca 720 actgacgaca atggcactac aaatgcagcc aagaagttgc ctccacagcagcttcctact 780 acaccaagcg cagacgctgg taacatatca agaattttta gccaagagaaacaatttgac 840 aactacttga acgaacggtt tatatttatg aaatggaagg aaaaatttttggtaccagat 900 gccctattaa tggaaggtgt agacggcgca tcttatgatg ggttttattacattgtccat 960 gatcaagtta ccgggaacat tcaagggttt tactatcatc aagatgctgaaaagttccaa 1020 cagctggaat tagtaccatc tttgaaaaat aaagtcgagt ccagtgattgttcttttgag 1080 tttgcttga 1089 73 822 PRT Saccharomyces cerevisiae 73Met Leu Glu Gly Thr Val Asp Tyr Asp Pro Leu Glu Asp Ile Thr Asn 1 5 1015 Ile Leu Phe Ser Lys Glu Ser Leu Asn Asn Ile Asp Glu Leu Ile Ser 20 2530 Ile Thr Arg Ser Tyr Lys Lys Gln Leu Gln Glu Asp Ile Leu Lys Glu 35 4045 Glu Asn Glu Leu Lys Glu His Pro Lys Asn Ser Ala Glu Ile Glu Ala 50 5560 Ser Leu Arg Lys Val Phe Gln Asp Phe Lys Glu Thr Gln Asp Val Ser 65 7075 80 Ala Ser Thr Glu Leu Thr Ile Ser Asn Leu Thr Glu Gly Ile Ser Tyr 8590 95 Leu Asp Ile Ala Lys Lys Asn Leu Thr His Ser Leu Thr Leu Phe Gln100 105 110 Asn Leu Lys Ile Leu Thr Asp Ser Tyr Ile Gln Cys Asn Glu LeuLeu 115 120 125 Ser Gln Gly Ser Phe Lys Lys Met Val Ser Pro Tyr Lys IleMet Cys 130 135 140 Ser Leu Ala Glu Asn Thr Phe Ile Ser Tyr Lys Ser LeuAsp Glu Ile 145 150 155 160 Asn Tyr Leu Leu Ser Ser Ile Ser Arg Leu LysGly Asp Thr Leu Ser 165 170 175 Lys Ile Lys Gln Asn Tyr Asn Ala Leu PheSer Gly Gly Asn Ile Ser 180 185 190 Glu His Asp Thr Ala Leu Thr Met GluLeu Arg Glu Gly Ala Cys Glu 195 200 205 Leu Leu Asp Cys Asp Thr Ser ThrArg Ala Gln Met Ile Asp Trp Cys 210 215 220 Leu Asp Lys Leu Leu Phe GluMet Lys Glu Ile Phe Arg Val Asp Asp 225 230 235 240 Glu Ala Gly Ser LeuGlu Asn Leu Ser Arg Arg Tyr Ile Tyr Phe Lys 245 250 255 Lys Ile Leu AsnAsn Phe Asn Ser Lys Phe Ala Asp Tyr Phe Leu Lys 260 265 270 Asp Trp GluMet Ala Val Arg Leu Thr Thr Thr Phe Tyr His Ile Thr 275 280 285 His LysAsp Leu Gln Thr Leu Leu Lys Arg Glu Phe Lys Asp Lys Asn 290 295 300 ProSer Ile Asp Leu Phe Met Thr Ala Leu Gln Ser Thr Leu Asp Phe 305 310 315320 Glu Lys Tyr Ile Asp Val Arg Phe Ser Lys Lys Ile Lys Glu Pro Lys 325330 335 Leu Ser Ser Cys Phe Glu Pro Tyr Leu Thr Leu Trp Val Ser His Gln340 345 350 Asn Gln Met Met Glu Lys Lys Phe Leu Ser Tyr Met Ser Glu ProLys 355 360 365 Tyr Pro Ser Asn Glu Thr Glu Ser Leu Val Leu Pro Ser SerAla Asp 370 375 380 Leu Phe Arg Thr Tyr Arg Ser Val Leu Thr Gln Thr LeuGlu Leu Ile 385 390 395 400 Asp Asn Asn Ala Asn Asp Ser Ile Leu Thr SerLeu Ala Asn Phe Phe 405 410 415 Ser Arg Trp Leu Gln Thr Tyr Ser Gln LysIle Leu Leu Pro Leu Leu 420 425 430 Leu Pro Asp Asn Ile Glu Val Gln AspLys Leu Glu Ala Ala Lys Tyr 435 440 445 Thr Val Leu Leu Ile Asn Thr AlaAsp Tyr Cys Ala Thr Thr Ile Asp 450 455 460 Gln Leu Glu Asp Lys Leu SerGlu Phe Ser Gly Asn Arg Glu Lys Leu 465 470 475 480 Ala Asn Ser Phe ThrLys Thr Lys Asn Ile Tyr Asp Asp Leu Leu Ala 485 490 495 Lys Gly Thr SerPhe Leu Leu Asn Arg Val Ile Pro Leu Asp Leu Asn 500 505 510 Phe Val TrpArg Glu Phe Ile Asn Asn Asp Trp Ser Asn Ala Ala Ile 515 520 525 Glu AspTyr Ser Arg Tyr Met Val Thr Leu Lys Ser Val Leu Lys Met 530 535 540 ProAla Leu Thr Asp Ala Ser Ile Lys Gln Gln Gln Glu Gln Pro Ser 545 550 555560 Thr Leu Ala Phe Ile Leu Ser Gln Phe Asn Arg Asp Val Tyr Lys Trp 565570 575 Asn Phe Leu Asp Lys Val Ile Asp Ile Ile Thr Thr Asn Phe Val Ser580 585 590 Asn Thr Ile Arg Leu Leu Gln Pro Val Pro Pro Phe Ser Leu AlaGly 595 600 605 Ser Lys Arg Lys Phe Glu Thr Arg Thr Val Val Asn Ile GlyGlu Gln 610 615 620 Leu Leu Leu Asp Leu Glu Leu Leu Lys Glu Ile Phe HisThr Leu Pro 625 630 635 640 Glu Ser Val Ser Asn Asp Ser Asp Leu Arg GluAsn Thr Ser Tyr Lys 645 650 655 Arg Val Lys Arg His Ala Asp Asn Asn IleAsp Gln Leu Leu Lys Phe 660 665 670 Ile Lys Leu Leu Met Ala Pro Leu AspSer Ala Asp Asp Tyr Tyr Glu 675 680 685 Thr Tyr Ser Lys Leu Thr Asn AsnAsn Pro Asp Ser Ala Val Trp Ser 690 695 700 Phe Val Leu Ala Leu Lys GlyIle Pro Trp Asp Leu Ala Leu Trp Lys 705 710 715 720 Lys Leu Trp Ser AlaTyr Asn Leu Glu Thr Asp Asp Thr Asp Glu Gly 725 730 735 Ser Arg Pro AspSer Asn Arg Asp Leu Phe Ile Phe Lys Trp Asp Lys 740 745 750 Val Leu LeuGly Gln Phe Glu Asn Asn Leu Ala Arg Met Gln Asp Pro 755 760 765 Asn TrpSer Lys Phe Val Arg Gln Asp Leu Lys Ile Ser Pro Pro Val 770 775 780 MetLys Arg Ile Val Ser Thr Pro Gln Ile Gln Gln Gln Lys Glu Glu 785 790 795800 Gln Lys Lys Gln Ser Leu Ser Val Lys Asp Phe Val Ser His Ser Arg 805810 815 Phe Phe Asn Arg Gly Thr 820 74 2000 DNA Saccharomyces cerevisiae74 atgctggaag gtacggtaga ttatgacccg ctggaagata ttaccaatat acttttttca 60aaagaatccc tgaacaacat agatgaactg atcagtatta ccagaagcta caaaaagcaa 120ttgcaagagg atattctcaa agaagagaat gaattgaagg aacaccctaa aaattccgct 180gaaatagagg cttctctgag gaaagttttc caagatttca aagaaactca agatgtctca 240gcctccaccg agttgacgat atcgaatctg acagaaggta tctcgtacct ggacattgcc 300aagaaaaacc tcacccactc tttgactctt ttccaaaatt taaagatatt gacagacagt 360tacatacaat gcaatgaatt actctcacag ggctcattca aaaaaatggt gtccccttat 420aagataatgt gttcgcttgc tgaaaacaca ttcatctctt acaaatcatt ggacgagata 480aactatttgt tgagctccat ttcaagactg aaaggagaca ctttgtccaa aattaaacaa 540aactacaatg cgctcttttc cggcggcaat atctcagagc atgatacagc actcactatg 600gaattgcgcg aaggtgcctg cgagctactc gactgcgata caagtacgag agcccagatg 660atagattggt gtttggacaa acttctcttc gaaatgaaag agatatttag ggtcgacgat 720gaagccggat ccctagaaaa tttatcgaga agatacattt acttcaaaaa aattcttaat 780aacttcaatt caaagttcgc agactatttc ttaaaagact gggaaatggc agtcagattg 840accaccactt tttatcacat tacacacaag gaccttcaga cacttctgaa aagggaattc 900aaagacaaga acccttccat tgatctattc atgacagcat tacaatcgac gctagatttc 960gaaaaataca tcgacgtacg attttcaaaa aaaattaagg aaccaaaact aagttcctgc 1020ttcgaacctt atttgacttt atgggtgtct caccaaaacc aaatgatgga aaagaaattt 1080ctttcttata tgagtgagcc gaagtaccca tctaatgaaa cagaatctct cgtgttaccc 1140tcgagtgcag accttttcag gacatatcgt tccgtactga ctcagacctt agagctcatt 1200gataataatg ccaatgatag catattgact tcattggcaa attttttcag tagatggctt 1260caaacttact cacaaaaaat tcttcttcct ttactgctgc ccgacaatat tgaagtccag 1320gataagctag aagctgccaa gtataccgtt ttattgatca atactgcaga ttattgtgcc 1380acgactatag atcaattgga ggataaatta tctgaattca gcggtaatcg tgaaaagctg 1440gcaaacagtt ttacgaaaac gaaaaatata tacgacgatt tactagcaaa aggaacttct 1500tttctattaa accgtgtcat acccttagat ctaaattttg tatggagaga gtttatcaac 1560aatgattggt caaatgctgc gatagaagat tatagcaggt acatggtaac cctcaaatcc 1620gtacttaaaa tgcccgcatt aacagatgcc tctattaaac aacagcaaga gcaaccttcg 1680actttggcat ttattttgtc gcaattcaat agagatgttt ataagtggaa tttcttggat 1740aaggtgattg atatcatcac tacaaatttt gtaagcaata ccatccgcct tctgcagccc 1800gttccaccct tttccctggc gggcagcaaa aggaaatttg aaaccagaac tgttgtcaac 1860attggcgagc agcttctcct tgatttagaa ttgctgaagg agatttttca cactttacca 1920gaaagtgtaa gtaacgattc tgacttgcga gaaaatacct cttataagag ggtgaaaaga 1980catgcagaca ataatataga 2000 75 779 PRT Saccharomyces cerevisiae 75 MetGlu Arg Thr Asn Thr Thr Thr Phe Lys Phe Phe Ser Leu Gly Gly 1 5 10 15Ser Asn Glu Val Gly Arg Ser Cys His Ile Leu Gln Tyr Lys Gly Lys 20 25 30Thr Val Met Leu Asp Ala Gly Ile His Pro Ala Tyr Gln Gly Leu Ala 35 40 45Ser Leu Pro Phe Tyr Asp Glu Phe Asp Leu Ser Lys Val Asp Ile Leu 50 55 60Leu Ile Ser His Phe His Leu Asp His Ala Ala Ser Leu Pro Tyr Val 65 70 7580 Met Gln Arg Thr Asn Phe Gln Gly Arg Val Phe Met Thr His Pro Thr 85 9095 Lys Ala Ile Tyr Arg Trp Leu Leu Arg Asp Phe Val Arg Val Thr Ser 100105 110 Ile Gly Ser Ser Ser Ser Ser Met Gly Thr Lys Asp Glu Gly Leu Phe115 120 125 Ser Asp Glu Asp Leu Val Asp Ser Phe Asp Lys Ile Glu Thr ValAsp 130 135 140 Tyr His Ser Thr Val Asp Val Asn Gly Ile Lys Phe Thr AlaPhe His 145 150 155 160 Ala Gly His Val Leu Gly Ala Ala Met Phe Gln IleGlu Ile Ala Gly 165 170 175 Leu Arg Val Leu Phe Thr Gly Asp Tyr Ser ArgGlu Val Asp Arg His 180 185 190 Leu Asn Ser Ala Glu Val Pro Pro Leu SerSer Asn Val Leu Ile Val 195 200 205 Glu Ser Thr Phe Gly Thr Ala Thr HisGlu Pro Arg Leu Asn Arg Glu 210 215 220 Arg Lys Leu Thr Gln Leu Ile HisSer Thr Val Met Arg Gly Gly Arg 225 230 235 240 Val Leu Leu Pro Val PheAla Leu Gly Arg Ala Gln Glu Ile Met Leu 245 250 255 Ile Leu Asp Glu TyrTrp Ser Gln His Ala Asp Glu Leu Gly Gly Gly 260 265 270 Gln Val Pro IlePhe Tyr Ala Ser Asn Leu Ala Lys Lys Cys Met Ser 275 280 285 Val Phe GlnThr Tyr Val Asn Met Met Asn Asp Asp Ile Arg Lys Lys 290 295 300 Phe ArgAsp Ser Gln Thr Asn Pro Phe Ile Phe Lys Asn Ile Ser Tyr 305 310 315 320Leu Arg Asn Leu Glu Asp Phe Gln Asp Phe Gly Pro Ser Val Met Leu 325 330335 Ala Ser Pro Gly Met Leu Gln Ser Gly Leu Ser Arg Asp Leu Leu Glu 340345 350 Arg Trp Cys Pro Glu Asp Lys Asn Leu Val Leu Ile Thr Gly Tyr Ser355 360 365 Ile Glu Gly Thr Met Ala Lys Phe Ile Met Leu Glu Pro Asp ThrIle 370 375 380 Pro Ser Ile Asn Asn Pro Glu Ile Thr Ile Pro Arg Arg CysGln Val 385 390 395 400 Glu Glu Ile Ser Phe Ala Ala His Val Asp Phe GlnGlu Asn Leu Glu 405 410 415 Phe Ile Glu Lys Ile Ser Ala Pro Asn Ile IleLeu Val His Gly Glu 420 425 430 Ala Asn Pro Met Gly Arg Leu Lys Ser AlaLeu Leu Ser Asn Phe Ala 435 440 445 Ser Leu Lys Gly Thr Asp Asn Glu ValHis Val Phe Asn Pro Arg Asn 450 455 460 Cys Val Glu Val Asp Leu Glu PheGln Gly Val Lys Val Ala Lys Ala 465 470 475 480 Val Gly Asn Ile Val AsnGlu Ile Tyr Lys Glu Glu Asn Val Glu Ile 485 490 495 Lys Glu Glu Ile AlaAla Lys Ile Glu Pro Ile Lys Glu Glu Asn Glu 500 505 510 Asp Asn Leu AspSer Gln Ala Glu Lys Gly Leu Val Asp Glu Glu Glu 515 520 525 His Lys AspIle Val Val Ser Gly Ile Leu Val Ser Asp Asp Lys Asn 530 535 540 Phe GluLeu Asp Phe Leu Ser Leu Ser Asp Leu Arg Glu His His Pro 545 550 555 560Asp Leu Ser Thr Thr Ile Leu Arg Glu Arg Gln Ser Val Arg Val Asn 565 570575 Cys Lys Lys Glu Leu Ile Tyr Trp His Ile Leu Gln Met Phe Gly Glu 580585 590 Ala Glu Val Leu Gln Asp Asp Asp Arg Val Thr Asn Gln Glu Pro Lys595 600 605 Val Lys Glu Glu Ser Lys Asp Asn Leu Thr Asn Thr Gly Lys LeuIle 610 615 620 Leu Gln Ile Met Gly Asp Ile Lys Leu Thr Ile Val Asn ThrLeu Ala 625 630 635 640 Val Val Glu Trp Thr Gln Asp Leu Met Asn Asp ThrVal Ala Asp Ser 645 650 655 Ile Ile Ala Ile Leu Met Asn Val Asp Ser AlaPro Ala Ser Val Lys 660 665 670 Leu Ser Ser His Ser Cys Asp Asp His AspHis Asn Asn Val Gln Ser 675 680 685 Asn Ala Gln Gly Lys Ile Asp Glu ValGlu Arg Val Lys Gln Ile Ser 690 695 700 Arg Leu Phe Lys Glu Gln Phe GlyAsp Cys Phe Thr Leu Phe Leu Asn 705 710 715 720 Lys Asp Glu Tyr Ala SerAsn Lys Glu Glu Thr Ile Thr Gly Val Val 725 730 735 Thr Ile Gly Lys SerThr Ala Lys Ile Asp Phe Asn Asn Met Lys Ile 740 745 750 Leu Glu Cys AsnSer Asn Pro Leu Lys Gly Arg Val Glu Ser Leu Leu 755 760 765 Asn Ile GlyGly Asn Leu Val Thr Pro Leu Cys 770 775 76 2000 DNA Saccharomycescerevisiae 76 atggagcgaa caaatacaac aacattcaaa tttttttcat tgggaggaagtaacgaagtt 60 ggacgatcat gtcatatatt gcaatataaa ggtaaaacag tgatgctcgacgcaggaatt 120 catccggcat atcaaggatt agcttcatta cctttttacg atgaatttgatctttccaaa 180 gtcgatatct tactgatatc acatttccat ttagaccatg ccgcttcacttccgtatgtg 240 atgcaacgga ctaactttca aggcagagtt ttcatgacac atccaaccaaagccatttac 300 agatggctac tgcgagattt tgtaagagtt actagtatag gttcttcatcctcctctatg 360 gggactaaag acgaaggtct attctcagat gaggatttag ttgattctttcgataaaatt 420 gaaacggtgg actatcattc tactgttgac gtcaatggta tcaaatttacggcattccat 480 gcaggccatg tattgggtgc agcgatgttt caaatagaga ttgctggccttagggtgtta 540 ttcacaggtg actattcgag agaagtagat cgtcacttaa attctgctgaggtgcctcca 600 ctttcatcca acgtattaat tgtggaatct acctttggta ccgctactcacgagccccgt 660 ttaaatagag aaaggaagct aacccaacta atccattcca cagtgatgagaggaggccgt 720 gttctactgc ctgtttttgc tttagggaga gctcaagaaa tcatgcttatactggatgag 780 tactggtctc aacatgctga tgaactcggt ggtggacaag tcccaatattttatgcatca 840 aatttggcaa aaaaatgtat gagcgttttt caaacctatg taaatatgatgaatgatgac 900 attagaaaaa aatttagaga ctctcagact aatcccttca tattcaaaaatatatcttat 960 cttagaaact tggaggattt ccaagatttt ggtcccagtg tgatgttggcctcaccaggt 1020 atgctgcaaa gtgggttatc aagagattta cttgaaaggt ggtgccctgaagataaaaat 1080 ctagtactaa tcaccggtta ctccatcgaa ggaacaatgg cgaaatttattatgcttgag 1140 ccagatacaa taccttccat aaataatccg gaaataacca ttccaagacgttgtcaagtt 1200 gaagaaatct cctttgccgc acacgttgac ttccaggaaa atttagaatttattgaaaag 1260 attagtgcgc caaatatcat ccttgttcat ggagaagcca atcccatgggccgtttgaaa 1320 tctgcattgt tatccaattt cgcgtcttta aagggtacag ataatgaagtccatgttttt 1380 aatcctagaa actgtgttga agtagatctt gaatttcaag gtgtcaaggttgcaaaagct 1440 gtgggaaata ttgtgaacga aatatataaa gaagaaaacg tagagataaaggaagaaatt 1500 gcggctaaaa ttgaacctat aaaggaagaa aacgaagaca acttggattctcaagcagaa 1560 aaaggtttgg ttgatgaaga ggaacacaaa gacatagtcg tttctgggattctagtttca 1620 gatgacaaaa atttcgaatt agacttcctt tctttgtctg atttaagagagcaccatccc 1680 gatctttcta caacgatatt aagagagcgc cagtcagttc gtgtaaattgtaaaaaggag 1740 ctaatttact ggcacatttt acaaatgttt ggagaggctg aagttcttcaagatgatgat 1800 agagtaacaa atcaagaacc aaaggttaaa gaagagtcaa aagacaatctgaccaataca 1860 ggtaaattga ttctacagat aatgggtgat attaagttaa ctattgttaatactctagcc 1920 gttgtggaat ggactcaaga tttaatgaat gacactgtag cagactccattattgctata 1980 cttatgaatg tggattcagc 2000 77 208 PRT Saccharomycescerevisiae 77 Met Ser Leu Ile His Pro Asp Thr Ala Lys Tyr Pro Phe LysPhe Glu 1 5 10 15 Pro Phe Leu Arg Gln Glu Tyr Ser Phe Ser Leu Asp ProAsp Arg Pro 20 25 30 Ile Cys Glu Phe Tyr Asn Ser Arg Glu Gly Pro Lys SerCys Pro Arg 35 40 45 Gly Pro Leu Cys Pro Lys Lys His Val Leu Pro Ile PheGln Asn Lys 50 55 60 Ile Val Cys Arg His Trp Leu Arg Gly Leu Cys Lys LysAsn Asp Gln 65 70 75 80 Cys Glu Tyr Leu His Glu Tyr Asn Leu Arg Lys MetPro Glu Cys Val 85 90 95 Phe Phe Ser Lys Asn Gly Tyr Cys Thr Gln Ser ProAsp Cys Gln Tyr 100 105 110 Leu His Ile Asp Pro Ala Ser Lys Ile Pro LysCys Glu Asn Tyr Glu 115 120 125 Met Gly Phe Cys Pro Leu Gly Ser Ser CysPro Arg Arg His Ile Lys 130 135 140 Lys Val Phe Cys Gln Arg Tyr Met ThrGly Phe Cys Pro Leu Gly Lys 145 150 155 160 Asp Glu Cys Asp Met Glu HisPro Gln Phe Ile Ile Pro Asp Glu Gly 165 170 175 Ser Lys Leu Arg Ile LysArg Asp Asp Glu Ile Asn Thr Arg Lys Met 180 185 190 Asp Glu Glu Lys GluArg Arg Leu Asn Ala Ile Ile Asn Gly Glu Val 195 200 205 78 627 DNASaccharomyces cerevisiae 78 atgagcctaa ttcaccccga tacagcaaaa tatccttttaaatttgaacc tttcctcagg 60 caagagtatt cgttttcact cgatcctgac agacctatttgtgaatttta caattctaga 120 gaaggcccta aatcatgtcc gaggggaccg ttatgtccaaaaaagcatgt gttaccaata 180 tttcagaata aaattgtttg tagacattgg cttcgagggttgtgcaaaaa gaatgaccaa 240 tgtgaatact tacatgaata caatcttcga aaaatgcctgaatgtgtctt cttcagcaaa 300 aacgggtact gtacacaaag tccagattgt caatatctacacatagatcc cgctagcaag 360 ataccaaaat gtgaaaatta cgaaatggga ttctgtcctctggggagttc ttgtcctaga 420 cggcatatta agaaggtttt ctgtcaaaga tacatgaccggattttgtcc tttagggaag 480 gatgaatgtg atatggaaca tccacagttc ataatcccagatgaaggtag taaattaaga 540 attaagagag acgatgagat aaataccagg aaaatggatgaagaaaagga aaggcgttta 600 aacgcaatta taaacggtga agtttga 627 79 107 PRTSaccharomyces cerevisiae 79 Met Val Lys Gly Lys Thr Phe Leu Lys Arg IleCys Pro Glu Glu Thr 1 5 10 15 Leu Asn Glu Glu Thr Lys Gln Glu Val SerVal Gly Phe Asp Lys Met 20 25 30 Arg Thr Leu Leu Arg Ser Arg Glu Ser GlyMet Thr Phe Ser Gln Gly 35 40 45 Pro Lys Leu Ala Ser Cys Gln Ser Val IleAsn Ala Ser Ser Glu Lys 50 55 60 Thr Ala Trp Thr Gln Leu Val Phe Arg LysSer Lys Met Lys Thr Tyr 65 70 75 80 Thr Lys Ser Val His Val Ile Phe IleAla Met Gly Glu Gly Glu Asp 85 90 95 Glu Ser Val Asp Met Asn Val Gly IleSer Tyr 100 105 80 324 DNA Saccharomyces cerevisiae 80 atggtaaagggtaaaacgtt tctgaaaaga atctgtccgg aagaaacgtt aaacgaagaa 60 actaagcaggaagtttcggt agggttcgat aagatgagaa ccctgttgcg gtctcgagaa 120 tcagggatgactttctccca aggacctaag ttagccagtt gccaatcagt gataaatgca 180 tcatctgaaaaaacggcttg gacacaactc gtgtttagga agagtaaaat gaagacgtac 240 accaagtctgtacacgttat cttcattgct atgggggaag gggaggatga aagtgttgat 300 atgaatgtaggtattagtta ttaa 324 81 425 PRT Saccharomyces cerevisiae 81 Met Thr AspPro Arg Arg Arg Thr Gly Arg His Phe Leu Thr Pro Glu 1 5 10 15 Asn LeuSer Ser Thr Leu Gln Ile Thr Asn Leu Pro Pro Glu Trp Asn 20 25 30 Gln AspIle Ile Thr Ser Val Val Ala Gly Ser Gly Pro Val Ile Asp 35 40 45 Ile LysAla Lys Asn Asp Pro Arg Thr Gly Lys Leu Thr Gly Val Leu 50 55 60 Phe AspTyr Leu Thr Ser Lys Asp Cys Lys Arg Ala Trp Glu Ile Leu 65 70 75 80 AsnArg Ile Glu Asn Phe Pro Val Lys Ile Glu Gln Ile Ile Pro Pro 85 90 95 AsnTyr Lys Asp His Leu Arg Glu Thr Ala Asn Lys Asn Ser Gln Lys 100 105 110Gln Val Leu Gln Leu Asn Arg Asp Ser Tyr Pro Phe Glu Ala Gly Leu 115 120125 Glu Leu Pro Phe Glu Met Val Thr Glu Val Pro Ile Pro Arg Arg Pro 130135 140 Pro Pro Pro Gln Ala Ala Asn Asn Thr Asn Ser Val Ser Asn Asn Thr145 150 155 160 Asn Ile Gln Phe Pro Asp Ile Leu Ser Lys Ala Ser Lys HisLeu Pro 165 170 175 Ser Phe Gln Asp Gly Ser Ile Ile Ala Pro Asp Lys IleSer Gln Asn 180 185 190 Leu Ser Lys Ile Pro Pro Leu Gln Leu Ile Glu IleIle Ser Asn Leu 195 200 205 Lys Ile Leu Ser Asn Gln Glu Asn Ile Gln LysSer Gln Leu Glu Ser 210 215 220 Phe Leu Asp Thr Asn Ser Asp Ile Thr IleSer Val Thr Gln Ala Leu 225 230 235 240 Leu Glu Met Gly Phe Ile Asp TyrSer Val Val Thr Lys Val Leu Lys 245 250 255 Ser Gln Val Gly Glu Ala ProSer Leu Leu Ser Ser Asn Asn Thr Ser 260 265 270 Asn Ser Asn Thr Pro ValSer Val Ile Arg Asn Asn Thr Pro Leu His 275 280 285 Val Pro Ser Asn GluVal Ser Asn Asn Pro Asn Asn Met Pro Leu Asn 290 295 300 Val Ala Met ProMet Pro Met Ser Thr Pro Pro Phe Ile Pro Leu Pro 305 310 315 320 Leu GlnGln Gln Pro Phe Gly Phe Ala Pro Pro Gly Pro Phe Met Pro 325 330 335 ProAla Gln Gly Pro Ser Met Gly Gln Pro Val Leu Ala Asn Gln Leu 340 345 350Gly Gln Val Gln Gln Gln Asn Ile Ser Ser Thr Glu Gly Pro Ser Asn 355 360365 Ala Asn Lys Ala Asn Asp Ser Gly Thr Ile Asn Met Ala Lys Leu Gln 370375 380 Leu Leu Pro Glu Asn Gln Gln Asp Met Ile Lys Gln Val Leu Thr Leu385 390 395 400 Thr Pro Ala Gln Ile Gln Ser Leu Pro Ser Asp Gln Gln LeuMet Val 405 410 415 Glu Asn Phe Arg Lys Glu Tyr Ile Ile 420 425 82 1278DNA Saccharomyces cerevisiae 82 atgacagatc ccagaagaag aacaggccgtcatttcttga caccggagaa tttatcctct 60 acattacaaa tcacaaactt acctccagaatggaaccaag atataattac ttcggtcgtg 120 gccggttctg gtccagttat agatataaaagctaagaatg acccgagaac tggtaaacta 180 accggtgtac tgttcgatta tttgactagtaaagattgta aacgcgcttg ggaaatttta 240 aatagaattg aaaactttcc cgtaaagatagagcaaataa tcccaccaaa ttataaggac 300 catcttagag aaacagcaaa taaaaattctcaaaagcagg tattacaact taatagagat 360 tcgtacccct tcgaggcggg tttggagctacctttcgaaa tggtgacaga agtccccatt 420 cctaggcgac caccgccacc acaggctgcaaataacacaa actctgtatc aaataacaca 480 aacattcaat tccccgacat actaagtaaagcatctaaac acttgccaag tttccaagat 540 ggctcgatta ttgcaccaga caaaatttcacaaaatttaa gtaaaattcc gccgttgcaa 600 cttattgaaa ttatatcaaa tttgaaaatattatcaaacc aagaaaacat ccaaaaatcg 660 caattagaat ctttcttaga tactaacagtgatatcacaa tatcagtgac ccaagcccta 720 ctagaaatgg gatttataga ctacagcgtggtgactaaag tgttgaaatc ccaagttggc 780 gaggccccat ctttgctttc gagtaataacacaagtaatt cgaacacccc cgtaagcgta 840 attagaaata acactccgtt gcatgtaccttctaatgaag tcagcaacaa tcctaacaat 900 atgccactga acgtagctat gccaatgcctatgtcgacac caccatttat ccctttacct 960 ctgcaacaac aaccgttcgg ttttgcgccaccgggccctt tcatgcctcc agctcaaggc 1020 ccctccatgg gacagcctgt gttggcaaatcaactcggcc aggtccagca acaaaatata 1080 agttctacag aaggaccctc taacgcgaataaagcgaatg acagcggcac cattaatatg 1140 gcgaaactgc aattactacc tgaaaaccaacaagatatga tcaaacaagt tcttactttg 1200 acacctgccc agatccaaag tttaccaagtgaccagcaac ttatggtgga aaactttaga 1260 aaagaatata taatctaa 1278 83 1592PRT Saccharomyces cerevisiae 83 Met Gly Thr Asp Pro Leu Ile Ile Arg AsnAsn Gly Ser Phe Trp Glu 1 5 10 15 Val Asp Asp Phe Thr Arg Leu Gly ArgThr Gln Leu Leu Ser Tyr Tyr 20 25 30 Leu Pro Leu Ala Ile Ile Ala Ser IleGly Ile Phe Ala Leu Cys Arg 35 40 45 Ser Gly Leu Ser Arg Tyr Val Arg SerAla Glu Cys Asp Leu Val Asn 50 55 60 Glu Tyr Leu Phe Gly Ala Gln Glu GluArg Lys Glu Asp Asn Ser Ile 65 70 75 80 Glu Arg Leu Leu Arg Asn Ser AsnThr Gln Ala Asn Tyr Val Asn Val 85 90 95 Lys Lys Gln Gly Arg Ile Leu LysLeu Arg His Phe Asp Ile Thr Thr 100 105 110 Ile Asp Val Lys Gln Ile AspAla Lys Asn His Gly Gly Leu Thr Phe 115 120 125 Ser Arg Pro Ser Thr SerAsp His Leu Arg Lys Ser Ser Glu Ile Val 130 135 140 Leu Met Ser Leu GlnIle Ile Gly Leu Ser Phe Leu Arg Val Thr Lys 145 150 155 160 Ile Asn IleGlu Leu Thr Asn Arg Asp Val Thr Thr Leu Leu Leu Phe 165 170 175 Trp LeuIle Leu Leu Ser Leu Ser Ile Leu Arg Val Tyr Lys Arg Ser 180 185 190 ThrAsn Leu Trp Ala Ile Cys Phe Thr Ala His Thr Thr Ile Trp Ile 195 200 205Ser Thr Trp Ile Pro Ile Arg Ser Val Tyr Ile Gly Asn Ile Asp Asp 210 215220 Val Pro Ser Gln Ile Phe Tyr Ile Phe Glu Phe Val Ile Thr Ser Thr 225230 235 240 Leu Gln Pro Ile Lys Leu Thr Ser Pro Ile Lys Asp Asn Ser SerIle 245 250 255 Ile Tyr Val Arg Asp Asp His Thr Ser Pro Ser Arg Glu HisIle Ser 260 265 270 Ser Ile Leu Ser Cys Ile Thr Trp Ser Trp Ile Thr AsnPhe Ile Trp 275 280 285 Glu Ala Gln Lys Asn Thr Ile Lys Leu Lys Asp IleTrp Gly Leu Ser 290 295 300 Met Glu Asp Tyr Ser Ile Phe Ile Leu Lys GlyPhe Thr Arg Arg Asn 305 310 315 320 Lys His Ile Asn Asn Leu Thr Leu AlaLeu Phe Glu Ser Phe Lys Thr 325 330 335 Tyr Leu Leu Ile Gly Met Leu TrpVal Leu Val Asn Ser Ile Val Asn 340 345 350 Leu Leu Pro Thr Ile Leu MetLys Arg Phe Leu Glu Ile Val Asp Asn 355 360 365 Pro Asn Arg Ser Ser SerCys Met Asn Leu Ala Trp Leu Tyr Ile Ile 370 375 380 Gly Met Phe Ile CysArg Leu Thr Leu Ala Ile Cys Asn Ser Gln Gly 385 390 395 400 Gln Phe ValSer Asp Lys Ile Cys Leu Arg Ile Arg Ala Ile Leu Ile 405 410 415 Gly GluIle Tyr Ala Lys Gly Leu Arg Arg Arg Leu Phe Thr Ser Pro 420 425 430 LysThr Ser Ser Asp Ser Asp Ser Ile Ser Ala Asn Leu Gly Thr Ile 435 440 445Ile Asn Leu Ile Ser Ile Asp Ser Phe Lys Val Ser Glu Leu Ala Asn 450 455460 Tyr Leu Tyr Val Thr Val Gln Ala Val Ile Met Ile Ile Val Val Val 465470 475 480 Gly Leu Leu Phe Asn Phe Leu Gly Val Ser Ala Phe Ala Gly IleSer 485 490 495 Ile Ile Leu Val Met Phe Pro Leu Asn Phe Leu Leu Ala AsnLeu Leu 500 505 510 Gly Lys Phe Gln Lys Gln Thr Leu Lys Cys Thr Asp GlnArg Ile Ser 515 520 525 Lys Leu Asn Glu Cys Leu Gln Asn Ile Arg Ile ValLys Tyr Phe Ala 530 535 540 Trp Glu Arg Asn Ile Ile Asn Glu Ile Lys SerIle Arg Gln Lys Glu 545 550 555 560 Leu Arg Ser Leu Leu Lys Lys Ser LeuVal Trp Ser Val Thr Ser Phe 565 570 575 Leu Trp Phe Val Thr Pro Thr LeuVal Thr Gly Val Thr Phe Ala Ile 580 585 590 Cys Thr Phe Val Gln His GluAsp Leu Asn Ala Pro Leu Ala Phe Thr 595 600 605 Thr Leu Ser Leu Phe ThrLeu Leu Lys Thr Pro Leu Asp Gln Leu Ser 610 615 620 Asn Met Leu Ser PheIle Asn Gln Ser Lys Val Ser Leu Lys Arg Ile 625 630 635 640 Ser Asp PheLeu Arg Met Asp Asp Thr Glu Lys Tyr Asn Gln Leu Thr 645 650 655 Ile SerPro Asp Lys Asn Lys Ile Glu Phe Lys Asn Ala Thr Leu Thr 660 665 670 TrpAsn Glu Asn Asp Ser Asp Met Asn Ala Phe Lys Leu Cys Gly Leu 675 680 685Asn Ile Lys Phe Gln Ile Gly Lys Leu Asn Leu Ile Leu Gly Ser Thr 690 695700 Gly Ser Gly Lys Ser Ala Leu Leu Leu Gly Leu Leu Gly Glu Leu Asn 705710 715 720 Leu Ile Ser Gly Ser Ile Ile Val Pro Ser Leu Glu Pro Lys HisAsp 725 730 735 Leu Ile Pro Asp Cys Glu Gly Leu Thr Asn Ser Phe Ala TyrCys Ser 740 745 750 Gln Ser Ala Trp Leu Leu Asn Asp Thr Val Lys Asn AsnIle Ile Phe 755 760 765 Asp Asn Phe Tyr Asn Glu Asp Arg Tyr Asn Lys ValIle Asp Ala Cys 770 775 780 Gly Leu Lys Arg Asp Leu Glu Ile Leu Pro AlaGly Asp Leu Thr Glu 785 790 795 800 Ile Gly Glu Lys Gly Ile Thr Leu SerGly Gly Gln Lys Gln Arg Ile 805 810 815 Ser Leu Ala Arg Ala Val Tyr SerSer Ala Lys His Val Leu Leu Asp 820 825 830 Asp Cys Leu Ser Ala Val AspSer His Thr Ala Val Trp Ile Tyr Glu 835 840 845 Asn Cys Ile Thr Gly ProLeu Met Lys Asn Arg Thr Cys Ile Leu Val 850 855 860 Thr His Asn Val SerLeu Thr Leu Arg Asn Ala His Phe Ala Ile Val 865 870 875 880 Leu Glu AsnGly Lys Val Lys Asn Gln Gly Thr Ile Thr Glu Leu Gln 885 890 895 Ser LysGly Leu Phe Lys Glu Lys Tyr Val Gln Leu Ser Ser Arg Asp 900 905 910 SerIle Asn Glu Lys Asn Ala Asn Arg Leu Lys Ala Pro Arg Lys Asn 915 920 925Asp Ser Gln Lys Ile Glu Pro Val Thr Glu Asn Ile Asn Phe Asp Ala 930 935940 Asn Phe Val Asn Asp Gly Gln Leu Ile Glu Glu Glu Glu Lys Ser Asn 945950 955 960 Gly Ala Ile Ser Pro Asp Val Tyr Lys Trp Tyr Leu Lys Phe PheGly 965 970 975 Gly Phe Lys Ala Leu Thr Ala Leu Phe Ala Leu Tyr Ile ThrAla Gln 980 985 990 Ile Leu Phe Ile Ser Gln Ser Trp Trp Ile Arg His TrpVal Asn Asp 995 1000 1005 Thr Asn Val Arg Ile Asn Ala Pro Gly Phe AlaMet Asp Thr Leu 1010 1015 1020 Pro Leu Lys Gly Met Thr Asp Ser Ser LysAsn Lys His Asn Ala 1025 1030 1035 Phe Tyr Tyr Leu Thr Val Tyr Phe LeuIle Gly Ile Ile Gln Ala 1040 1045 1050 Met Leu Gly Gly Phe Lys Thr MetMet Thr Phe Leu Ser Gly Met 1055 1060 1065 Arg Ala Ser Arg Lys Ile PheAsn Asn Leu Leu Asp Leu Val Leu 1070 1075 1080 His Ala Gln Ile Arg PhePhe Asp Val Thr Pro Val Gly Arg Ile 1085 1090 1095 Met Asn Arg Phe SerLys Asp Ile Glu Gly Val Asp Gln Glu Leu 1100 1105 1110 Ile Pro Tyr LeuGlu Val Thr Ile Phe Cys Leu Ile Gln Cys Ala 1115 1120 1125 Ser Ile IlePhe Leu Ile Thr Val Ile Thr Pro Arg Phe Leu Thr 1130 1135 1140 Val AlaVal Ile Val Phe Val Leu Tyr Phe Phe Val Gly Lys Trp 1145 1150 1155 TyrLeu Thr Ala Ser Arg Glu Leu Lys Arg Leu Asp Ser Ile Thr 1160 1165 1170Lys Ser Pro Ile Phe Gln His Phe Ser Glu Thr Leu Val Gly Val 1175 11801185 Cys Thr Ile Arg Ala Phe Gly Asp Glu Arg Arg Phe Ile Leu Glu 11901195 1200 Asn Met Asn Lys Ile Asp Gln Asn Asn Arg Ala Phe Phe Tyr Leu1205 1210 1215 Ser Val Thr Val Lys Trp Phe Ser Phe Arg Val Asp Met IleGly 1220 1225 1230 Ala Phe Ile Val Leu Ala Ser Gly Ser Phe Ile Leu LeuAsn Ile 1235 1240 1245 Ala Asn Ile Asp Ser Gly Leu Ala Gly Ile Ser LeuThr Tyr Ala 1250 1255 1260 Ile Leu Phe Thr Asp Gly Ala Leu Trp Leu ValArg Leu Tyr Ser 1265 1270 1275 Thr Phe Glu Met Asn Met Asn Ser Val GluArg Leu Lys Glu Tyr 1280 1285 1290 Ser Ser Ile Glu Gln Glu Asn Tyr LeuGly His Asp Glu Gly Arg 1295 1300 1305 Ile Leu Leu Leu Asn Glu Pro SerTrp Pro Lys Asp Gly Glu Ile 1310 1315 1320 Glu Ile Glu Asn Leu Ser LeuArg Tyr Ala Pro Asn Leu Pro Pro 1325 1330 1335 Val Ile Arg Asn Val SerPhe Lys Val Asp Pro Gln Ser Lys Ile 1340 1345 1350 Gly Ile Val Gly ArgThr Gly Ala Gly Lys Ser Thr Ile Ile Thr 1355 1360 1365 Ala Leu Phe ArgLeu Leu Glu Pro Ile Thr Gly Cys Ile Lys Ile 1370 1375 1380 Asp Gly GlnAsp Ile Ser Lys Ile Asp Leu Val Thr Leu Arg Arg 1385 1390 1395 Ser IleThr Ile Ile Pro Gln Asp Pro Ile Leu Phe Ala Gly Thr 1400 1405 1410 IleLys Ser Asn Val Asp Pro Tyr Asp Glu Tyr Asp Glu Lys Lys 1415 1420 1425Ile Phe Lys Ala Leu Ser Gln Val Asn Leu Ile Ser Ser His Glu 1430 14351440 Phe Glu Glu Val Leu Asn Ser Glu Glu Arg Phe Asn Ser Thr His 14451450 1455 Asn Lys Phe Leu Asn Leu His Thr Glu Ile Ala Glu Gly Gly Leu1460 1465 1470 Asn Leu Ser Gln Gly Glu Arg Gln Leu Leu Phe Ile Ala ArgSer 1475 1480 1485 Leu Leu Arg Glu Pro Lys Ile Ile Leu Leu Asp Glu AlaThr Ser 1490 1495 1500 Ser Ile Asp Tyr Asp Ser Asp His Leu Ile Gln GlyIle Ile Arg 1505 1510 1515 Ser Glu Phe Asn Lys Ser Thr Ile Leu Thr IleAla His Arg Leu 1520 1525 1530 Arg Ser Val Ile Asp Tyr Asp Arg Ile IleVal Met Asp Ala Gly 1535 1540 1545 Glu Val Lys Glu Tyr Asp Arg Pro SerGlu Leu Leu Lys Asp Glu 1550 1555 1560 Arg Gly Ile Phe Tyr Ser Met CysArg Asp Ser Gly Gly Leu Glu 1565 1570 1575 Leu Leu Lys Gln Ile Ala LysGln Ser Ser Lys Met Met Lys 1580 1585 1590 84 2000 DNA Saccharomycescerevisiae 84 atgggaacgg atccccttat tatccgaaat aatggttcat tttgggaagttgatgatttt 60 actcgtttag gaagaactca gctattgagc tactatttac cattggctatcatagcctca 120 attggcattt tcgcactttg tcgcagtgga ttatctcgtt atgtaagatctgccgagtgc 180 gatttagtga acgaatatct atttggcgca caagaagaga gaaaagaagataatagtata 240 gaaagacttc tacggaactc aaatacccaa gccaattacg tcaacgtcaaaaagcaagga 300 aggattttga aacttagaca ttttgatata acaactatag atgtcaagcaaatcgatgct 360 aaaaatcatg gtggactaac gtttagtaga ccgtctacta gtgaccacttaagaaaatca 420 tctgaaattg tattaatgtc tttacaaata attggccttt cctttttaagagtaacaaaa 480 atcaatattg aattaacgaa cagagatgtt acaactttac tattattttggttaatacta 540 ctttccctaa gtatcttaag agtttacaag cgttcaacga atctttgggccatctgtttt 600 actgcccata caactatttg gatttcaacc tggattccaa ttcgttcggtctatattggt 660 aatatcgatg atgtaccctc acagatattt tacatctttg aattcgtaattacttcaacc 720 ttacagccaa taaagctcac ttcaccgatt aaagacaact catccatcatctacgttaga 780 gacgaccata cgtctccttc gagggaacac atatcctcaa ttttaagttgcattacttgg 840 agctggatta ccaattttat atgggaggcc caaaagaaca ctattaagttaaaggatatt 900 tggggcttat caatggaaga ctatagcatt ttcattctaa aagggtttaccaggagaaac 960 aagcacatta ataatttgac gctagcactg tttgaatctt tcaaaacatatttactcata 1020 ggaatgttat gggttctggt gaacagtatt gtaaaccttc ttcccacaattttaatgaaa 1080 agatttttag aaattgtgga taacccaaac cgttcctcat catgcatgaatttggcgtgg 1140 ctttatatta ttggtatgtt catttgtaga ttgacattag caatttgtaattcccaaggt 1200 caatttgttt ctgataagat ttgtttaaga ataagagcca tactcataggagaaatttat 1260 gcaaaaggct tacgtaggag gctgtttaca tctccaaaaa ccagctctgattcagatagt 1320 atctccgcaa accttggtac cataattaat ctcatttcta ttgactcatttaaggtatcg 1380 gaactagcaa actaccttta tgtgacagtt caggcagtaa ttatgataatagttgttgta 1440 ggactacttt tcaacttttt aggtgtttca gcttttgcag gaatttcaattatcttagtg 1500 atgttcccat tgaatttctt gttagcgaat ttgttaggta agtttcaaaagcaaacactg 1560 aaatgtactg accaaagaat ctcaaaattg aacgagtgct tacagaacataagaattgtc 1620 aaatattttg cttgggaaag gaatattata aatgaaatca aatcaataaggcaaaaggaa 1680 ttaagatcct tattaaaaaa atctttggtg tggtccgtaa cttcttttctttggttcgtg 1740 acaccgacct tggtgacagg tgtcactttc gccatctgta catttgttcaacatgaagat 1800 ttgaatgccc cgcttgcttt cactactttg tcactcttca ctttgttaaagacacccctg 1860 gatcaattat caaatatgct aagtttcata aatcaatcaa aagtctctctaaaaagaata 1920 agcgattttt taaggatgga cgatacagaa aaatataatc aactaaccatatctccagac 1980 aaaaataaaa ttgaatttaa 2000 85 329 PRT Saccharomycescerevisiae 85 Met Thr Thr Val Ser Ile Asn Lys Pro Asn Leu Leu Lys PheLys His 1 5 10 15 Val Lys Ser Phe Gln Pro Gln Glu Lys Asp Cys Gly ProVal Thr Ser 20 25 30 Leu Asn Phe Asp Asp Asn Gly Gln Phe Leu Leu Thr SerSer Ser Asn 35 40 45 Asp Thr Met Gln Leu Tyr Ser Ala Thr Asn Cys Lys PheLeu Asp Thr 50 55 60 Ile Ala Ser Lys Lys Tyr Gly Cys His Ser Ala Ile PheThr His Ala 65 70 75 80 Gln Asn Glu Cys Ile Tyr Ser Ser Thr Met Lys AsnPhe Asp Ile Lys 85 90 95 Tyr Leu Asn Leu Glu Thr Asn Gln Tyr Leu Arg TyrPhe Ser Gly His 100 105 110 Gly Ala Leu Val Asn Asp Leu Lys Met Asn ProVal Asn Asp Thr Phe 115 120 125 Leu Ser Ser Ser Tyr Asp Glu Ser Val ArgLeu Trp Asp Leu Lys Ile 130 135 140 Ser Lys Pro Gln Val Ile Ile Pro SerLeu Val Pro Asn Cys Ile Ala 145 150 155 160 Tyr Asp Pro Ser Gly Leu ValPhe Ala Leu Gly Asn Pro Glu Asn Phe 165 170 175 Glu Ile Gly Leu Tyr AsnLeu Lys Lys Ile Gln Glu Gly Pro Phe Leu 180 185 190 Ile Ile Lys Ile AsnAsp Ala Thr Phe Ser Gln Trp Asn Lys Leu Glu 195 200 205 Phe Ser Asn AsnGly Lys Tyr Leu Leu Val Gly Ser Ser Ile Gly Lys 210 215 220 His Leu IlePhe Asp Ala Phe Thr Gly Gln Gln Leu Phe Glu Leu Ile 225 230 235 240 GlyThr Arg Ala Phe Pro Met Arg Glu Phe Leu Asp Ser Gly Ser Ala 245 250 255Cys Phe Thr Pro Asp Gly Glu Phe Val Leu Gly Thr Asp Tyr Asp Gly 260 265270 Arg Ile Ala Ile Trp Asn His Ser Asp Ser Ile Ser Asn Lys Val Leu 275280 285 Arg Pro Gln Gly Phe Ile Pro Cys Val Ser His Glu Thr Cys Pro Arg290 295 300 Ser Ile Ala Phe Asn Pro Lys Tyr Ser Met Phe Val Thr Ala AspGlu 305 310 315 320 Thr Val Asp Phe Tyr Val Tyr Asp Glu 325 86 990 DNASaccharomyces cerevisiae 86 atgaccaccg tgtccatcaa taagcccaac ctgctgaaattcaagcatgt taaaagcttt 60 caacctcaag aaaaagactg cggacccgta acctcattgaatttcgacga taatggccag 120 tttctactga cctcttcttc caacgataca atgcaattgtacagtgccac gaactgcaaa 180 ttcttggaca ctatagcctc taagaaatat ggctgtcactccgctatctt tacgcacgca 240 caaaacgaat gtatctattc ctctacaatg aaaaattttgacattaaata ccttaatctg 300 gaaacaaacc aatatctaag atatttttcc ggtcatggcgccctagtgaa tgatttgaag 360 atgaaccccg tgaacgatac gtttctatcg tcgtcatacgatgaatccgt taggctttgg 420 gatttgaaga tctctaaacc gcaagttatt ataccaagtctcgtaccaaa ttgtatcgca 480 tatgatccaa gtggccttgt attcgcattg gggaacccagagaatttcga aatagggcta 540 tataatctga aaaaaattca ggagggtcct ttcttgataattaaaattaa tgatgcgact 600 ttcagtcaat ggaataaatt agaattttct aacaatggaaagtatttatt agttggctcc 660 tcgataggaa agcatttaat ttttgacgca ttcacaggtcaacaattatt cgaactaata 720 ggaacaaggg ccttcccgat gagagaattt ctagattctggatctgcttg tttcacacca 780 gatggtgaat tcgtccttgg aaccgattat gacggtaggattgccatttg gaatcattct 840 gattcaataa gtaacaaagt attaaggccg caagggttcattccctgtgt ttctcatgag 900 acctgcccca ggtcaattgc attcaaccct aaatattcgatgtttgttac cgcagacgaa 960 acagtagatt tttacgtgta cgatgaatga 990 87 220PRT Saccharomyces cerevisiae 87 Met Ser Ala Gly Asp Ile Ser Ala Ile AsnIle Lys Ser Val Lys Lys 1 5 10 15 Asn Arg Arg Arg Lys Lys Arg Arg ThrAla Asp Val Ser Ser Ser Asp 20 25 30 Ser Ser Ser Ser Asp Pro Ser Ser GluSer Glu Lys Glu Glu Ile Gln 35 40 45 Asn Gly Ala Ile Glu Glu His Val GlyGlu Asn Gly Lys Ser Asp His 50 55 60 Val Phe Ser Lys Gly Asn Asp Glu AspLys Gln Glu Asp Ile Ala Ile 65 70 75 80 Glu Val Ser Asp Val Glu Leu ThrAsp Glu Glu Ser Lys Asp Leu Lys 85 90 95 Leu Asn Ser Lys Glu Val Ile AspAsp Leu Thr Lys Ile Ser Leu Ser 100 105 110 Lys Ile Pro Glu Pro Thr LysSer Gln Asn Lys Glu Gly Phe Met Asn 115 120 125 Ala Ser Lys Ile Ala GluAsn Ile Lys Leu Ala Arg Glu Glu Tyr Asn 130 135 140 Glu Leu Ala Glu AsnPhe Val Pro Lys Gly Lys Asp Lys Thr Lys Leu 145 150 155 160 Arg Glu GluTyr Leu Asn Leu Leu Phe Glu Asn Tyr Gly Asp Asp Ile 165 170 175 Asn ArgLeu Arg Ala Ala Pro Asp Phe Thr Asn Lys Ser Leu Ser Ile 180 185 190 LeuAla Asp Ala Leu Gln Glu Gly Ile Gly Met Phe Asp Ile Gly Glu 195 200 205Leu Glu Leu Val Leu Lys Asn Lys Glu Met Glu Asn 210 215 220 88 663 DNASaccharomyces cerevisiae 88 atgtcggcag gtgatatatc agccataaat atcaagtctgtcaaaaaaaa cagaaggagg 60 aagaagagaa gaacagctga tgtttcatca tcagattcttcatcatcgga tccatcatca 120 gaaagtgaaa aggaggaaat ccaaaatggg gccatcgaagaacacgttgg agaaaatggt 180 aaaagtgatc atgttttctc aaaaggtaat gacgaagacaaacaagagga cattgcaata 240 gaagtttcgg atgtcgagct tacagacgaa gaaagcaaggatttgaagtt aaattcaaaa 300 gaagtgatag acgatttaac caaaatttct ttgagcaagatcccagagcc tacgaaatct 360 caaaacaagg agggttttat gaatgcatcg aaaattgccgaaaatatcaa gcttgcgaga 420 gaagaataca atgaattggc agaaaacttt gtgcccaaagggaaagacaa gacaaagtta 480 agggaagaat acttaaattt actttttgaa aactacggtgatgatatcaa tcgtcttaga 540 gctgccccgg atttcacgaa taaatcacta tccattttggcagatgctct gcaggaaggc 600 ataggaatgt ttgatattgg tgaactagaa ttggtcttgaaaaataaaga aatggagaac 660 tga 663 89 441 PRT Saccharomyces cerevisiae 89Met Ser Ser Thr Ile Phe Tyr Arg Phe Lys Ser Gln Arg Asn Thr Ser 1 5 1015 Arg Ile Leu Phe Asp Gly Thr Gly Leu Thr Val Phe Asp Leu Lys Arg 20 2530 Glu Ile Ile Gln Glu Asn Lys Leu Gly Asp Gly Thr Asp Phe Gln Leu 35 4045 Lys Ile Tyr Asn Pro Asp Thr Glu Glu Glu Tyr Asp Asp Asp Ala Phe 50 5560 Val Ile Pro Arg Ser Thr Ser Val Ile Val Lys Arg Ser Pro Ala Ile 65 7075 80 Lys Ser Phe Ser Val His Ser Arg Leu Lys Gly Asn Val Gly Ala Ala 8590 95 Ala Leu Gly Asn Ala Thr Arg Tyr Val Thr Gly Arg Pro Arg Val Leu100 105 110 Gln Lys Arg Gln His Thr Ala Thr Thr Thr Ala Asn Val Ser GlyThr 115 120 125 Thr Glu Glu Glu Arg Ile Ala Ser Met Phe Ala Thr Gln GluAsn Gln 130 135 140 Trp Glu Gln Thr Gln Glu Glu Met Ser Ala Ala Thr ProVal Phe Phe 145 150 155 160 Lys Ser Gln Thr Asn Lys Asn Ser Ala Gln GluAsn Glu Gly Pro Pro 165 170 175 Pro Pro Gly Tyr Met Cys Tyr Arg Cys GlyGly Arg Asp His Trp Ile 180 185 190 Lys Asn Cys Pro Thr Asn Ser Asp ProAsn Phe Glu Gly Lys Arg Ile 195 200 205 Arg Arg Thr Thr Gly Ile Pro LysLys Phe Leu Lys Ser Ile Glu Ile 210 215 220 Asp Pro Glu Thr Met Thr ProGlu Glu Met Ala Gln Arg Lys Ile Met 225 230 235 240 Ile Thr Asp Glu GlyLys Phe Val Val Gln Val Glu Asp Lys Gln Ser 245 250 255 Trp Glu Asp TyrGln Arg Lys Arg Glu Asn Arg Gln Ile Asp Gly Asp 260 265 270 Glu Thr IleTrp Arg Lys Gly His Phe Lys Asp Leu Pro Asp Asp Leu 275 280 285 Lys CysPro Leu Thr Gly Gly Leu Leu Arg Gln Pro Val Lys Thr Ser 290 295 300 LysCys Cys Asn Ile Asp Phe Ser Lys Glu Ala Leu Glu Asn Ala Leu 305 310 315320 Val Glu Ser Asp Phe Val Cys Pro Asn Cys Glu Thr Arg Asp Ile Leu 325330 335 Leu Asp Ser Leu Val Pro Asp Gln Asp Lys Glu Lys Glu Val Glu Thr340 345 350 Phe Leu Lys Lys Gln Glu Glu Leu His Gly Ser Ser Lys Asp GlyAsn 355 360 365 Gln Pro Glu Thr Lys Lys Met Lys Leu Met Asp Pro Thr GlyThr Ala 370 375 380 Gly Leu Asn Asn Asn Thr Ser Leu Pro Thr Ser Val AsnAsn Gly Gly 385 390 395 400 Thr Pro Val Pro Pro Val Pro Leu Pro Phe GlyIle Pro Pro Phe Pro 405 410 415 Met Phe Pro Met Pro Phe Met Pro Pro ThrAla Thr Ile Thr Asn Pro 420 425 430 His Gln Ala Asp Ala Ser Pro Lys Lys435 440 90 1326 DNA Saccharomyces cerevisiae 90 atgagtagca cgatattttaccgctttaag tctcaacgaa acacatcaag aattttattt 60 gatggtaccg gcctgacagtatttgatttg aaaagggaaa ttattcaaga gaacaaacta 120 ggtgacggca cagatttccaattaaaaatt tacaacccag atacagaaga ggaatacgac 180 gatgatgcct ttgttatacctagatctact agtgtcatag taaaaagatc tccagcaatt 240 aaatcattct ccgtacacagtcgacttaaa gggaatgtgg gagcagcagc tcttgggaac 300 gcaacaaggt atgttactggtaggccaaga gtgttgcaaa agagacaaca cactgctaca 360 accactgcta atgttagtggtacaacggaa gaagaaagaa ttgctagtat gtttgccaca 420 caagaaaatc aatgggaacaaacgcaagaa gaaatgtctg cagccacacc tgtttttttc 480 aagtcacaga cgaataagaattctgcacaa gaaaacgaag gcccaccgcc accaggttat 540 atgtgctatc gttgtgggggtagagaccac tggattaaaa attgtccaac taacagcgat 600 ccaaattttg aaggaaaaagaatcagaaga accacaggta ttccaaagaa gtttttaaaa 660 tccattgaaa tagatcccgagacaatgaca ccggaagaga tggctcagcg aaagattatg 720 attacggacg aaggcaagttcgtggtacaa gttgaagaca aacaatcatg ggaagactac 780 caaaggaaaa gagagaaccgtcaaattgat ggtgatgaaa ccatttggag aaaaggccat 840 ttcaaagatc ttcctgacgatttaaaatgt cccttgacag gtggtctttt gaggcagccg 900 gtaaagacaa gcaagtgctgtaacatagat ttctcaaaag aggcgctgga aaatgcactg 960 gtagagagcg actttgtatgccccaattgc gaaacccgcg atatccttct cgattcttta 1020 gtacccgacc aggacaaggaaaaggaggtc gaaacgtttt tgaagaaaca agaggaacta 1080 cacggaagct ctaaagatggcaaccagcca gaaactaaga aaatgaagtt gatggatcca 1140 actggcaccg ctggcttgaacaacaatacc agccttccaa cttctgtaaa taacggcggt 1200 acgccagtgc caccagtaccgttacctttc ggtatacctc ctttccccat gtttccaatg 1260 cccttcatgc ctccaacggctactatcaca aatcctcatc aagctgacgc aagccctaag 1320 aaatga 1326 91 159 PRTSaccharomyces cerevisiae 91 Met Ser Arg Met Pro Ser Ser Phe Asp Val ThrGlu Arg Asp Leu Asp 1 5 10 15 Asp Met Thr Phe Gly Glu Arg Ile Ile TyrHis Cys Lys Lys Gln Pro 20 25 30 Leu Val Pro Ile Gly Cys Leu Leu Thr ThrGly Ala Val Ile Leu Ala 35 40 45 Ala Gln Asn Val Arg Leu Gly Asn Lys TrpLys Ala Gln Tyr Tyr Phe 50 55 60 Arg Trp Arg Val Gly Leu Gln Ala Ala ThrLeu Val Ala Leu Val Ala 65 70 75 80 Gly Ser Phe Ile Tyr Gly Thr Ser GlyLys Glu Leu Lys Ala Lys Glu 85 90 95 Glu Gln Leu Lys Glu Lys Ala Lys MetArg Glu Lys Leu Trp Ile Gln 100 105 110 Glu Leu Glu Arg Arg Glu Glu GluThr Glu Ala Arg Arg Lys Arg Ala 115 120 125 Glu Leu Ala Arg Met Lys ThrLeu Glu Asn Glu Glu Glu Ile Lys Asn 130 135 140 Leu Glu Lys Glu Leu SerAsp Leu Glu Asn Lys Leu Gly Lys Lys 145 150 155 92 480 DNA Saccharomycescerevisiae 92 atgtcacgca tgccatctag tttcgatgtt acggagaggg atttggatgatatgaccttt 60 ggcgaaagga ttatatacca ttgtaagaaa cagccattgg tacccattgggtgcttgctg 120 actacaggag ctgtcattct ggctgctcaa aatgttcgtc ttggtaataaatggaaagct 180 cagtactact tccgttggcg tgtgggtcta caagcggcca cactagtcgcactagtcgca 240 ggttcattta tctatgggac ttctggtaag gaactgaagg cgaaggaggaacaattgaag 300 gagaaagcca agatgagaga aaagttatgg atccaagagc tggagagaagggaggaagaa 360 acggaggcaa ggagaaaaag agccgaattg gcaagaatga agacccttgagaacgaagag 420 gaaatcaaga acttagaaaa ggaactaagc gacctggaaa ataagcttggaaagaagtaa 480 93 188 PRT Saccharomyces cerevisiae 93 Met Asp Leu ProLys Asp Lys Ser Asp Arg Thr His Gln Arg Ile Asn 1 5 10 15 Leu Asn AsnSer Gly Thr Asp Arg Thr Asn Asp Leu Tyr Leu His Ile 20 25 30 Val Gln ThrPhe Gly Cys Ile Glu Thr Thr Ala Thr Glu Asn Ala Thr 35 40 45 Lys Leu LeuMet Leu Gly Asp Val Glu Val Glu Ile Ser Ala Ser Ser 50 55 60 Val Ser IleGlu Trp Thr Gln Lys Ser Met Ile Ser Gln Thr Ile Ala 65 70 75 80 Asp SerIle Val Ile Met Ile Ile Gly Leu Cys Ala Ser Asp Lys Asn 85 90 95 Val LeuSer Glu Ser Glu Leu Lys Glu Arg Asn His Asn Val Trp Lys 100 105 110 IleGln Glu Leu Gln Asn Leu Phe Arg Glu Gln Phe Gly Asp Ser Phe 115 120 125Ser Ile Asp Glu Gly Ile Gly Lys Lys Glu Asn Val Lys Asn Gly Ser 130 135140 Val Thr Ile Gly Lys Ser Lys Ala Thr Ile Asp Phe Ser Thr Met Lys 145150 155 160 Leu Ile Asp Cys Asn Ser Asn Pro Leu Lys Gly Arg Val Glu SerIle 165 170 175 Leu Ser Ile Gly Gln Lys Leu Thr Thr Pro Leu Cys 180 18594 567 DNA Saccharomyces cerevisiae 94 atggatttac ccaaggacaa aagtgacagaactcatcaaa gaattaatct aaataacagt 60 gggacagatc gaactaatga tttgtaccttcatattgtcc aaacgttcgg ttgcatagaa 120 acaactgcaa cggaaaatgc cacgaaactgttaatgctgg gtgacgtcga agtagaaata 180 tctgcgagca gcgtttcaat tgagtggacacagaagtcaa tgataagcca aacaattgcc 240 gatagtatag taataatgat catcggtttgtgtgcaagcg acaagaacgt gctatctgaa 300 tcagaattga aagagagaaa ccataacgtttggaagatcc aagaattgca aaatctgttt 360 cgagaacaat ttggagacag ttttagcatcgatgaaggaa taggaaaaaa agaaaatgta 420 aagaatggta gcgtcaccat aggcaagagtaaagccacga tcgatttctc caccatgaag 480 ctgattgatt gtaattcgaa cccactaaagggaagagtgg agagcatact aagcattggc 540 cagaaattaa caactccatt gtgctga 567

1. An isolated complex selected from complex (I) and comprising (a) afirst protein, or a functionally active fragment or functionally activederivative thereof, which first protein is selected from the groupconsisting of: (i) Cft1 (SEQ ID NO:3), or a mammalian homolog thereof,or a variant of Cft1 encoded by a nucleic acid that hybridizes to theCft1 nucleic acid (SEQ ID NO:4) or its complement under low stringencyconditions, (ii) Cft2 (SEQ ID NO:5), or a mammalian homolog thereof, ora variant of Cft2 encoded by a nucleic acid that hybridizes to the Cft2nucleic acid (SEQ ID NO:6) or its complement under low stringencyconditions, (iii) Clp1 (SEQ ID NO:9), or a mammalian homolog thereof, ora variant of Clp1 encoded by a nucleic acid that hybridizes to the Clp1nucleic acid (SEQ ID NO:10) or its complement under low stringencyconditions, (iv) Fip1 (SEQ ID NO:19), or a mammalian homolog thereof, ora variant of Fip1 encoded by a nucleic acid that hybridizes to the Fip1nucleic acid (SEQ ID NO:20) or its complement under low stringencyconditions, (v) Pab1 (SEQ ID NO:33), or a mammalian homolog thereof, ora variant of Pab1 encoded by a nucleic acid that hybridizes to the Pab1nucleic acid (SEQ ID NO:34) or its complement under low stringencyconditions, (vi) Pap1 (SEQ ID NO:35), or a mammalian homolog thereof, ora variant of Pap1 encoded by a nucleic acid that hybridizes to the Pap1nucleic acid (SEQ ID NO:36) or its complement under low stringencyconditions, (vii) Pcf11 (SEQ ID NO:37), or a mammalian homolog thereof,or a variant of Pcf11 encoded by a nucleic acid that hybridizes to thePcf11 nucleic acid (SEQ ID NO:38) or its complement under low stringencyconditions, (viii) Pfs2 (SEQ ID NO:43), or a mammalian homolog thereof,or a variant of Pfs2 encoded by a nucleic acid that hybridizes to thePfs2 nucleic acid (SEQ ID NO:44) or its complement under low stringencyconditions, (ix) Pta1 (SEQ ID NO:45), or a mammalian homolog thereof, ora variant of Pta1 encoded by a nucleic acid that hybridizes to the Pta1nucleic acid (SEQ ID NO:46) or its complement under low stringencyconditions, (x) Rna14 (SEQ ID NO:49), or a mammalian homolog thereof, ora variant of Rna14 encoded by a nucleic acid that hybridizes to theRna14 nucleic acid (SEQ ID NO:50) or its complement under low stringencyconditions, (xi) Rna15 (SEQ ID NO:51), or a mammalian homolog thereof,or a variant of Rna15 encoded by a nucleic acid that hybridizes to theRna15 nucleic acid (SEQ ID NO:52) or its complement under low stringencyconditions, (xii) Tif4632 (SEQ ID NO:63), or a mammalian homologthereof, or a variant of Tif4632 encoded by a nucleic acid thathybridizes to the Tif4632 nucleic acid (SEQ ID NO:64) or its complementunder low stringency conditions, (xiii) Ykl059c (SEQ ID NO:89), or amammalian homolog thereof, or a variant of Ykl059c encoded by a nucleicacid that hybridizes to the Ykl059c nucleic acid (SEQ ID NO:90) or itscomplement under low stringency conditions, (xiv) Ysh1 (SEQ ID NO:75),or a mammalian homolog thereof, or a variant of Ysh1 encoded by anucleic acid that hybridizes to the Ysh1 nucleic acid (SEQ ID NO:76) orits complement under low stringency conditions, and (xv) Yth1 (SEQ IDNO:77), or a mammalian homolog thereof, or a variant of Yth1 encoded bya nucleic acid that hybridizes to the Yth1 nucleic acid (SEQ ID NO:78)or its complement under low stringency conditions; and (b) a secondprotein, or a functionally active fragment or functionally activederivative thereof, which second protein is selected from the groupconsisting of: (i) Act1 (SEQ ID NO:1), or a mammalian homolog thereof,or a variant of Act1 encoded by a nucleic acid that hybridizes to theAct1 nucleic acid (SEQ ID NO:2) or its complement under low stringencyconditions, (ii) Cka1 (SEQ ID NO:7), or a mammalian homolog thereof, ora variant of Cka1 encoded by a nucleic acid that hybridizes to the Cka1nucleic acid (SEQ ID NO:8) or its complement under low stringencyconditions, (iii) Eft2 (SEQ ID NO:11), or a mammalian homolog thereof,or a variant of Eft2 encoded by a nucleic acid that hybridizes to theEft2 nucleic acid (SEQ ID NO:12) or its complement under low stringencyconditions, (iv) Eno2 (SEQ ID NO:13), or a mammalian homolog thereof, ora variant of Eno2 encoded by a nucleic acid that hybridizes to the Eno2nucleic acid (SEQ ID NO:14) or its complement under low stringencyconditions, (v) Glc7 (SEQ ID NO:15), or a mammalian homolog thereof, ora variant of Glc7 encoded by a nucleic acid that hybridizes to the Glc7nucleic acid (SEQ ID NO:16) or its complement under low stringencyconditions, (vi) Gpm1 (SEQ ID NO:17), or a mammalian homolog thereof, ora variant of Gpm1 encoded by a nucleic acid that hybridizes to the Gpm1nucleic acid (SEQ ID NO:18) or its complement under low stringencyconditions, (vii) Hhf2 (SEQ ID NO:21), or a mammalian homolog thereof,or a variant of Hhf2 encoded by a nucleic acid that hybridizes to theHhf2 nucleic acid (SEQ ID NO:22) or its complement under low stringencyconditions, (viii) Hta1 (SEQ ID NO:23), or a mammalian homolog thereof,or a variant of Hta1 encoded by a nucleic acid that hybridizes to theHta1 nucleic acid (SEQ ID NO:24) or its complement under low stringencyconditions, (ix) Hsc82 (SEQ ID NO:25), or a mammalian homolog thereof,or a variant of Hsc82 encoded by a nucleic acid that hybridizes to theHsc82 nucleic acid (SEQ ID NO:26) or its complement under low stringencyconditions, (x) Imd2 (SEQ ID NO:27), or a mammalian homolog thereof, ora variant of Imd2 encoded by a nucleic acid that hybridizes to the Imd2nucleic acid (SEQ ID NO:28) or its complement under low stringencyconditions, (xi) Imd4 (SEQ ID NO:29), or a mammalian homolog thereof, ora variant of Imd4 encoded by a nucleic acid that hybridizes to the Imd4nucleic acid (SEQ ID NO:30) or its complement under low stringencyconditions, (xii) Met6 (SEQ ID NO:31), or a mammalian homolog thereof,or a variant of Met6 encoded by a nucleic acid that hybridizes to theMet6 nucleic acid (SEQ ID NO:32) or its complement under low stringencyconditions, (xiii) Pdc1 (SEQ ID NO:39), or a mammalian homolog thereof,or a variant of Pdc1 encoded by a nucleic acid that hybridizes to thePdc1 nucleic acid (SEQ ID NO:40) or its complement under low stringencyconditions, (xiv) Pfk1 (SEQ ID NO:41), or a mammalian homolog thereof,or a variant of Pfk1 encoded by a nucleic acid that hybridizes to thePfk1 nucleic acid (SEQ ID NO:42) or its complement under low stringencyconditions, (xv) Ref2 (SEQ ID NO:47), or a mammalian homolog thereof, ora variant of Ref2 encoded by a nucleic acid that hybridizes to the Ref2nucleic acid (SEQ ID NO:48) or its complement under low stringencyconditions, (xvi) Sec13 (SEQ ID NO:53), or a mammalian homolog thereof,or a variant of Sec13 encoded by a nucleic acid that hybridizes to theSec13 nucleic acid (SEQ ID NO:54) or its complement under low stringencyconditions, (xvii) Sec31 (SEQ ID NO:55), or a mammalian homolog thereof,or a variant of Sec31 encoded by a nucleic acid that hybridizes to theSec31 nucleic acid (SEQ ID NO:56) or its complement under low stringencyconditions, (xviii) Ssa3 (SEQ ID NO:57), or a mammalian homolog thereof,or a variant of Ssa3 encoded by a nucleic acid that hybridizes to theSsa3 nucleic acid (SEQ ID NO:58) or its complement under low stringencyconditions, (xix) Ssu72 (SEQ ID NO:59), or a mammalian homolog thereof,or a variant of Ssu72 encoded by a nucleic acid that hybridizes to theSsu72 nucleic acid (SEQ ID NO:60) or its complement under low stringencyconditions, (xx) Taf60 (SEQ ID NO:61), or a mammalian homolog thereof,or a variant of Taf60 encoded by a nucleic acid that hybridizes to theTaf60 nucleic acid (SEQ ID NO:62) or its complement under low stringencyconditions, (xxi) Tkl1 (SEQ ID NO:65), or a mammalian homolog thereof,or a variant of Tkl1 encoded by a nucleic acid that hybridizes to theTkl1 nucleic acid (SEQ ID NO:66) or its complement under low stringencyconditions, (xxii) Tsa1 (SEQ ID NO:67), or a mammalian homolog thereof,or a variant of Tsa1 encoded by a nucleic acid that hybridizes to theTsa1 nucleic acid (SEQ ID NO:68) or its complement under low stringencyconditions, (xxiii) Tye7 (SEQ ID NO:69), or a mammalian homolog thereof,or a variant of Tye7 encoded by a nucleic acid that hybridizes to theTye7 nucleic acid (SEQ ID NO:70) or its complement under low stringencyconditions, (xxiv) Vid24 (SEQ ID NO:71), or a mammalian homolog thereof,or a variant of Vid24 encoded by a nucleic acid that hybridizes to theVid24 nucleic acid (SEQ ID NO:72) or its complement under low stringencyconditions, (xxv) Vps53 (SEQ ID NO:73), or a mammalian homolog thereof,or a variant of Vps53 encoded by a nucleic acid that hybridizes to theVps53 nucleic acid (SEQ ID NO:74) or its complement under low stringencyconditions, (xxvi) Ycl046w (SEQ ID NO:79), or a mammalian homologthereof, or a variant of Ycl046w encoded by a nucleic acid thathybridizes to the Ycl046w nucleic acid (SEQ ID NO:80) or its complementunder low stringency conditions, (xxvii) Ygr156w (SEQ ID NO:81), or amammalian homolog thereof, or a variant of Ygr156w encoded by a nucleicacid that hybridizes to the Ygr156w nucleic acid (SEQ ID NO:82) or itscomplement under low stringency conditions, (xxviii) Yhl035c (SEQ IDNO:83), or a mammalian homolog thereof, or a variant of Yhl035c encodedby a nucleic acid that hybridizes to the Yhl035c nucleic acid (SEQ IDNO:84) or-its complement under low stringency conditions, (xxix) Ykl018w(SEQ ID NO:85), or a mammalian homolog thereof, or a variant of Ykl018wencoded by a nucleic acid that hybridizes to the Ykl018w nucleic acid(SEQ ID NO:86) or its complement under low stringency conditions, (xxx)Ylr221c (SEQ ID NO:87), or a mammalian homolog thereof, or a variant ofYlr221c encoded by a nucleic acid that hybridizes to the Ylr221c nucleicacid (SEQ ID NO:88) or its complement under low stringency conditions,(xxxi) Yml030w (SEQ ID NO:91), or a mammalian homolog thereof, or avariant of Yml030w encoded by a nucleic acid that hybridizes to theYml030w nucleic acid (SEQ ID NO:92) or its complement under lowstringency conditions, and (xxxii) Yor179c (SEQ ID NO:93), or amammalian homolog thereof, or a variant of Yor179c encoded by a nucleicacid that hybridizes to the Yor179c nucleic acid (SEQ ID NO:94) or itscomplement under low stringency conditions, wherein said first proteinand said second protein are members of a native cellularPolyadenylation-complex, and wherein said low stringency conditionscomprise hybridization in a buffer comprising 35% formamide, 5×SSC, 50mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 55° C., andwashing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS for 1.5 hours at 60° C., and a complex (II)comprising at least two second proteins.
 2. An isolated complexcomprising the following proteins: (i) Act1 (SEQ ID NO:1), or amammalian homolog thereof, or a variant of Act1 encoded by a nucleicacid that hybridizes to the Act1 nucleic acid (SEQ ID NO:2) or itscomplement under low stringency conditions, (ii) Cft1 (SEQ ID NO:3), ora mammalian homolog thereof, or a variant of Cft1 encoded by a nucleicacid that hybridizes to the Cft1 nucleic acid (SEQ ID NO:4) or itscomplement under low stringency conditions, (iii) Cft2 (SEQ ID NO:5), ora mammalian homolog thereof, or a variant of Cft2 encoded by a nucleicacid that hybridizes to the Cft2 nucleic acid (SEQ ID NO:6) or itscomplement under low stringency conditions, (iv) Cka1 (SEQ ID NO:7), ora mammalian homolog thereof, or a variant of Cka1 encoded by a nucleicacid that hybridizes to the Cka1 nucleic acid (SEQ ID NO:8) or itscomplement under low stringency conditions, (v) Clp1 (SEQ ID NO:9), or amammalian homolog thereof, or a variant of Clp1 encoded by a nucleicacid that hybridizes to the Clp1 nucleic acid (SEQ ID NO:10) or itscomplement under low stringency conditions, (vi) Eft2 (SEQ ID NO:11), ora mammalian homolog thereof, or a variant of Eft2 encoded by a nucleicacid that hybridizes to the Eft2 nucleic acid (SEQ ID NO:12) or itscomplement under low stringency conditions, (vii) Eno2 (SEQ ID NO:13),or a mammalian homolog thereof, or a variant of Eno2 encoded by anucleic acid that hybridizes to the Eno2 nucleic acid (SEQ ID NO:14) orits complement under low stringency conditions, (viii) Glc7 (SEQ IDNO:15), or a mammalian homolog thereof, or a variant of Glc7 encoded bya nucleic acid that hybridizes to the Glc7 nucleic acid (SEQ ID NO:16)or its complement under low stringency conditions, (ix) Gpm1 (SEQ IDNO:17), or a mammalian homolog thereof, or a variant of Gpm1 encoded bya nucleic acid that hybridizes to the Gpm1 nucleic acid (SEQ ID NO:18)or its complement under low stringency conditions, (x) Fip1 (SEQ IDNO:19), or a mammalian homolog thereof, or a variant of Fip1 encoded bya nucleic acid that hybridizes to the Fip1 nucleic acid (SEQ ID NO:20)or its complement under low stringency conditions, (xi) Hhf2 (SEQ IDNO:21), or a mammalian homolog thereof, or a variant of Hhf2 encoded bya nucleic acid that hybridizes to the Hhf2 nucleic acid (SEQ ID NO:22)or its complement under low stringency conditions, (xii) Hta1 (SEQ IDNO:23), or a mammalian homolog thereof, or a variant of Hta1 encoded bya nucleic acid that hybridizes to the Hta1 nucleic acid (SEQ ID NO:24)or its complement under low stringency conditions, (xiii) Hsc82 (SEQ IDNO:25), or a mammalian homolog thereof, or a variant of Hsc82 encoded bya nucleic acid that hybridizes to the Hsc82 nucleic acid (SEQ ID NO:26)or its complement under low stringency conditions, (xiv) Imd2 (SEQ IDNO:27), or a mammalian homolog thereof, or a variant of Imd2 encoded bya nucleic acid that hybridizes to the Imd2 nucleic acid (SEQ ID NO:28)or its complement under low stringency conditions, (xv) Imd4 (SEQ IDNO:29), or a mammalian homolog thereof, or a variant of Imd4 encoded bya nucleic acid that hybridizes to the Imd4 nucleic acid (SEQ ID NO:30)or its complement under low stringency conditions, (xvi) Met6 (SEQ IDNO:31), or a mammalian homolog thereof, or a variant of Met6 encoded bya nucleic acid that hybridizes to the Met6 nucleic acid (SEQ ID NO:32)or its complement under low stringency conditions, (xvii) Pab1 (SEQ IDNO:33), or a mammalian homolog thereof, or a variant of Pab1 encoded bya nucleic acid that hybridizes to the Pab1 nucleic acid (SEQ ID NO:34)or its complement under low stringency conditions, (xviii) Pap1 (SEQ IDNO:35), or a mammalian homolog thereof, or a variant of Pap1 encoded bya nucleic acid that hybridizes to the Pap1 nucleic acid (SEQ ID NO:36)or its complement under low stringency conditions, (xix) Pcf11 (SEQ IDNO:37), or a mammalian homolog thereof, or a variant of Pcf11 encoded bya nucleic acid that hybridizes to the Pcf11 nucleic acid (SEQ ID NO:38)or its complement under low stringency conditions, (xx) Pdc1 (SEQ IDNO:39), or a mammalian homolog thereof, or a variant of Pdc1 encoded bya nucleic acid that hybridizes to the Pdc1 nucleic acid (SEQ ID NO:40)or its complement under low stringency conditions, (xxi) Pfk1 (SEQ IDNO:41), or a mammalian homolog thereof, or a variant of Pfk1 encoded bya nucleic acid that hybridizes to the Pfk1 nucleic acid (SEQ ID NO:42)or its complement under low stringency conditions, (xxii) Pfs2 (SEQ IDNO:43), or a mammalian homolog thereof, or a variant of Pfs2 encoded bya nucleic acid that hybridizes to the Pfs2 nucleic acid (SEQ ID NO:44)or its complement under low stringency conditions, (xxiii) Pta1 (SEQ IDNO:45), or a mammalian homolog thereof, or a variant of Pta1 encoded bya nucleic acid that hybridizes to the Pta1 nucleic acid (SEQ ID NO:46)or its complement under low stringency conditions, (xxiv) Ref2 (SEQ IDNO:47), or a mammalian homolog thereof, or a variant of Ref2 encoded bya nucleic acid that hybridizes to the Ref2 nucleic acid (SEQ ID NO:48)or its complement under low stringency conditions, (xxv) Rna14 (SEQ IDNO:49), or a mammalian homolog thereof, or a variant of Rna14 encoded bya nucleic acid that hybridizes to the Rna14 nucleic acid (SEQ ID NO:50)or its complement under low stringency conditions, (xxvi) Rna15 (SEQ IDNO:51), or a mammalian homolog thereof, or a variant of Rna15 encoded bya nucleic acid that hybridizes to the Rna15 nucleic acid (SEQ ID NO:52)or its complement under low stringency conditions, (xxvii) Sec13 (SEQ IDNO:53), or a mammalian homolog thereof, or a variant of Sec13 encoded bya nucleic acid that hybridizes to the Sec13 nucleic acid (SEQ ID NO:54)or its complement under low stringency conditions, (xxviii) Sec31 (SEQID NO:55), or a mammalian homolog thereof, or a variant of Ser31 encodedby a nucleic acid that hybridizes to the Sec31 nucleic acid (SEQ IDNO:56) or its complement under low stringency conditions, (xxix) Ssa3(SEQ ID NO:57), or a mammalian homolog thereof, or a variant of Ssa3encoded by a nucleic acid that hybridizes to the Ssa3 nucleic acid (SEQID NO:58) or its complement under low stringency conditions, (xxx) Ssu72(SEQ ID NO:59), or a mammalian homolog thereof, or a variant of Ssu72encoded by a nucleic acid that hybridizes to the Ssu72 nucleic acid (SEQID NO:60) or its complement under low stringency conditions, (xxxi)Taf60 (SEQ ID NO:61), or a mammalian homolog thereof, or a variant ofTaf60 encoded by a nucleic acid that hybridizes to the Taf60 nucleicacid (SEQ ID NO:62) or its complement under low stringency conditions,(xxxii) Tif4632 (SEQ ID NO:63), or a mammalian homolog thereof, or avariant of Tif4632 encoded by a nucleic acid that hybridizes to theTif4632 nucleic acid (SEQ ID NO:64) or its complement under lowstringency conditions, (xxxiii) Tkl1 (SEQ ID NO:65), or a mammalianhomolog thereof, or a variant of Tkl1 encoded by a nucleic acid thathybridizes to the Tkl1 nucleic acid (SEQ ID NO:66) or its complementunder low stringency conditions, (xxxiv) Tsa1 (SEQ ID NO:67), or amammalian homolog thereof, or a variant of Tsa1 encoded by a nucleicacid that hybridizes to the Tsa1 nucleic acid (SEQ ID NO:68) or itscomplement under low stringency conditions, (xxxv) Tye7 (SEQ ID NO:69),or a mammalian homolog thereof, or a variant of Tye7 encoded by anucleic acid that hybridizes to the Tye7 nucleic acid (SEQ ID NO:70) orits complement under low stringency conditions, (xxxvi) Vid24 (SEQ IDNO:71), or a mammalian homolog thereof, or a variant of Vid24 encoded bya nucleic acid that hybridizes to the Vid24 nucleic acid (SEQ ID NO:72)or its complement under low stringency conditions, (xxxvii) Vps53 (SEQID NO:73), or a mammalian homolog thereof, or a variant of Vps53 encodedby a nucleic acid that hybridizes to the Vps53 nucleic acid (SEQ IDNO:74) or its complement under low stringency conditions, (xxxviii) Ysh1(SEQ ID NO:75), or a mammalian homolog thereof, or a variant of Ysh1encoded by a nucleic acid that hybridizes to the Ysh1 nucleic acid (SEQID NO:76) or its complement under low stringency conditions, (xxxix)Yth1 (SEQ ID NO:77), or a mammalian homolog thereof, or a variant ofYth1 encoded by a nucleic acid that hybridizes to the Yth1 nucleic acid(SEQ ID NO:78) or its complement under low stringency conditions, (xl)Ycl046w (SEQ ID NO:79), or a mammalian homolog thereof, or a variant ofYcl046w encoded by a nucleic acid that hybridizes to the Ycl046w nucleicacid (SEQ ID NO:80) or its complement under low stringency conditions,(xli) Ygr156w (SEQ ID NO:81), or a mammalian homolog thereof, or avariant of Ygr156w encoded by a nucleic acid that hybridizes to theYgr156w nucleic acid (SEQ ID NO:82) or its complement under lowstringency conditions, (xlii) Yhl035c (SEQ ID NO:83), or a mammalianhomolog thereof, or a variant of Yhl035c encoded by a nucleic acid thathybridizes to the Yhl035c nucleic acid (SEQ ID NO:84) or its complementunder low stringency conditions, (xliii) Ykl018w (SEQ ID NO:85), or amammalian homolog thereof, or a variant of Ykl018w encoded by a nucleicacid that hybridizes to the Ykl018w nucleic acid (SEQ ID NO:86) or itscomplement under low stringency conditions, (xliv) Ylr221c (SEQ IDNO:87), or a mammalian homolog thereof, or a variant of Ylr221c encodedby a nucleic acid that hybridizes to the Ylr221c nucleic acid (SEQ IDNO:88) or its complement under low stringency conditions, (xlv) Ykl059c(SEQ ID NO:89), or a mammalian homolog thereof, or a variant of Ykl059cencoded by a nucleic acid that hybridizes to the Ykl059c nucleic acid(SEQ ID NO:90) or its complement under low stringency conditions, (xlvi)Yml030w (SEQ ID NO:91), or a mammalian homolog thereof, or a variant ofYml030w encoded by a nucleic acid that hybridizes to the Yml030w nucleicacid (SEQ ID NO:92) or its complement under low stringency conditions,and (xlvii) Yor179c (SEQ ID NO:93), or a mammalian homolog thereof, or avariant of Yor179c encoded by a nucleic acid that hybridizes to theYor179c nucleic acid (SEQ ID NO:94) or its complement under lowstringency conditions, wherein said proteins are members of a nativecellular Polyadenylation-complex, and wherein said low stringencyconditions comprise hybridization in a buffer comprising 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2%BSA, 100 ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextransulfate for 18-20 hours at 40° C., washing in a buffer consisting of2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at55° C., and washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60° C.
 3. An isolatedcomplex that comprises all but 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17,18,19,20,21,22,23,24,25,26,27 or 28 of the following 47proteins: (i) Act1 (SEQ ID NO:1), or a mammalian homolog thereof, or avariant of Act1 encoded by a nucleic acid that hybridizes to the Act1nucleic acid (SEQ ID NO:2) or its complement under low stringencyconditions, (ii) Cft1 (SEQ ID NO:3), or a mammalian homolog thereof, ora variant of Cft1 encoded by a nucleic acid that hybridizes to the Cft1nucleic acid (SEQ ID NO-4) or its complement under low stringencyconditions, (iii) Cft2 (SEQ ID NO:5), or a mammalian homolog thereof, ora variant of Cft2 encoded by a nucleic acid that hybridizes to the Cft2nucleic acid (SEQ ID NO:6) or its complement under low stringencyconditions, (iv) Cka1 (SEQ ID NO:7), or a mammalian homolog thereof, ora variant of Cka1 encoded by a nucleic acid that hybridizes to the Cka1nucleic acid (SEQ ID NO:8) or its complement under low stringencyconditions, (v) Clp1 (SEQ ID NO:9), or a mammalian homolog thereof, or avariant of Clp1 encoded by a nucleic acid that hybridizes to the Clp1nucleic acid (SEQ ID NO:10) or its complement under low stringencyconditions, (vi) Eft2 (SEQ ID NO:11), or a mammalian homolog thereof, ora variant of Eft2 encoded by a nucleic acid that hybridizes to the Eft2nucleic acid (SEQ ID NO:12) or its complement under low stringencyconditions, (vii) Eno2 (SEQ ID NO:13), or a mammalian homolog thereof,or a variant of Eno2 encoded by a nucleic acid that hybridizes to theEno2 nucleic acid (SEQ ID NO:14) or its complement under low stringencyconditions, (viii) Glc7 (SEQ ID NO:15), or a mammalian homolog thereof,or a variant of Glc7 encoded by a nucleic acid that hybridizes to theGlc7 nucleic acid (SEQ ID NO:16) or its complement under low stringencyconditions, (ix) Gpm1 (SEQ ID NO:17), or a mammalian homolog thereof, ora variant of Gpm1 encoded by a nucleic acid that hybridizes to the Gpm1nucleic acid (SEQ ID NO:18) or its complement under low stringencyconditions, (x) Fip1 (SEQ ID NO:19), or a mammalian homolog thereof, ora variant of Fip1 encoded by a nucleic acid that hybridizes to the Fip1nucleic acid (SEQ ID NO:20) or its complement under low stringencyconditions, (xi) Hhf2 (SEQ ID NO:21), or a mammalian homolog thereof, ora variant of Hhf2 encoded by a nucleic acid that hybridizes to the Hhf2nucleic acid (SEQ ID NO:22) or its complement under low stringencyconditions, (xii) Hta1 (SEQ ID NO:23), or a mammalian homolog thereof,or a variant of Hta1 encoded by a nucleic acid that hybridizes to theHta1 nucleic acid (SEQ ID NO:24) or its complement under low stringencyconditions, (xiii) Hsc82 (SEQ ID NO:25), or a mammalian homolog thereof,or a variant of Hsc82 encoded by a nucleic acid that hybridizes to theHsc82 nucleic acid (SEQ ID NO:26) or its complement under low stringencyconditions, (xiv) Imd2 (SEQ ID NO:27), or a mammalian homolog thereof,or a variant of Imd2 encoded by a nucleic acid that hybridizes to theImd2 nucleic acid (SEQ ID NO:28) or its complement under low stringencyconditions, (xv) Imd4 (SEQ ID NO:29), or a mammalian homolog thereof, ora variant of Imd4 encoded by a nucleic acid that hybridizes to the Imd4nucleic acid (SEQ ID NO:30) or its complement under low stringencyconditions, (xvi) Met6 (SEQ ID NO:31), or a mammalian homolog thereof,or a variant of Met6 encoded by a nucleic acid that hybridizes to theMet6 nucleic acid (SEQ ID NO:32) or its complement under low stringencyconditions, (xvii) Pab1 (SEQ ID NO:33), or a mammalian homolog thereof,or a variant of Pab1 encoded by a nucleic acid that hybridizes to thePab1 nucleic acid (SEQ ID NO:34) or its complement under low stringencyconditions, (xviii) Pap1 (SEQ ID NO:35), or a mammalian homolog thereof,or a variant of Pap1 encoded by a nucleic acid that hybridizes to thePap1 nucleic acid (SEQ ID NO:36) or its complement under low stringencyconditions, (xix) Pcf11 (SEQ ID NO:37), or a mammalian homolog thereof,or a variant of Pcf11 encoded by a nucleic acid that hybridizes to thePcf11 nucleic acid (SEQ ID NO:38) or its complement under low stringencyconditions, (xx) Pdc1 (SEQ ID NO:39), or a mammalian homolog thereof, ora variant of Pdc1 encoded by a nucleic acid that hybridizes to the Pdc1nucleic acid (SEQ ID NO:40) or its complement under low stringencyconditions, (xxi) Pfk1 (SEQ ID NO:41), or a mammalian homolog thereof,or a variant of Pfk1 encoded by a nucleic acid that hybridizes to thePfk1 nucleic acid (SEQ ID NO:42) or its complement under low stringencyconditions, (xxii) Pfs2 (SEQ ID NO:43), or a mammalian homolog thereof,or a variant of Pfs2 encoded by a nucleic acid that hybridizes to thePfs2 nucleic acid (SEQ ID NO:44) or its complement under low stringencyconditions, (xxiii) Pta1 (SEQ ID NO:45), or a mammalian homolog thereof,or a variant of Pta1 encoded by a nucleic acid that hybridizes to thePta1 nucleic acid (SEQ ID NO:46) or its complement under low stringencyconditions, (xxiv) Ref2 (SEQ ID NO:47), or a mammalian homolog thereof,or a variant of Ref2 encoded by a nucleic acid that hybridizes to theRef2 nucleic acid (SEQ ID NO:48) or its complement under low stringencyconditions, (xxv) Rna14 (SEQ ID NO:49), or a mammalian homolog thereof,or a variant of Rna14 encoded by a nucleic acid that hybridizes to theRna14 nucleic acid (SEQ ID NO:50) or its complement under low stringencyconditions, (xxvi) Rna15 (SEQ ID NO:51), or a mammalian homolog thereof,or a variant of Rna15 encoded by a nucleic acid that hybridizes to theRna15 nucleic acid (SEQ ID NO:52) or its complement under low stringencyconditions, (xxvii) Sec13 (SEQ ID NO:53), or a mammalian homologthereof, or a variant of Sec13 encoded by a nucleic acid that hybridizesto the Sec13 nucleic acid (SEQ ID NO:54) or its complement under lowstringency conditions, (xxviii) Sec31 (SEQ ID NO:55), or a mammalianhomolog thereof, or a variant of Sec31 encoded by a nucleic acid thathybridizes to the Sec31 nucleic acid (SEQ ID NO:56) or its complementunder low stringency conditions, (xxix) Ssa3 (SEQ ID NO:57), or amammalian homolog thereof, or a variant of Ssa3 encoded by a nucleicacid that hybridizes to the Ssa3 nucleic acid (SEQ ID NO:58) or itscomplement under low stringency conditions, (xxx) Ssu72 (SEQ ID NO:59),or a mammalian homolog thereof, or a variant of Ssu72 encoded by anucleic acid that hybridizes to the Ssu72 nucleic acid (SEQ ID NO:60) orits complement under low stringency conditions, (xxxi) Taf60 (SEQ IDNO:61), or a mammalian homolog thereof, or a variant of Taf60 encoded bya nucleic acid that hybridizes to the Taf60 nucleic acid (SEQ ID NO:62)or its complement under low stringency conditions, (xxxii) Tif4632 (SEQID NO:63), or a mammalian homolog thereof, or a variant of Tif4632encoded by a nucleic acid that hybridizes to the Tif4632 nucleic acid(SEQ ID NO:64) or its complement under low stringency conditions,(xxxiii) Tkl1 (SEQ ID NO:65), or a mammalian homolog thereof, or avariant of Tkl1 encoded by a nucleic acid that hybridizes to the Tkl1nucleic acid (SEQ ID NO:66) or its complement under low stringencyconditions, (xxxiv) Tsa1 (SEQ ID NO:67), or a mammalian homolog thereof,or a variant of Tsa1 encoded by a nucleic acid that hybridizes to theTsa1 nucleic acid (SEQ ID NO:68) or its complement under low stringencyconditions, (xxxv) Tye7 (SEQ ID NO:69), or a mammalian homolog thereof,or a variant of Tye7 encoded by a nucleic acid that hybridizes to theTye7 nucleic acid (SEQ ID NO:70) or its complement under low stringencyconditions, (xxxvi) Vid24 (SEQ ID NO:71), or a mammalian homologthereof, or a variant of Vid24 encoded by a nucleic acid that hybridizesto the Vid24 nucleic acid (SEQ ID NO:72) or its complement under lowstringency conditions, (xxxvii) Vps53 (SEQ ID NO:73), or a mammalianhomolog thereof, or a variant of Vps53 encoded by a nucleic acid thathybridizes to the Vps53 nucleic acid (SEQ ID NO:74) or its complementunder low stringency conditions, (xxxviii) Ysh1 (SEQ ID NO:75), or amammalian homolog thereof, or a variant of Ysh1 encoded by a nucleicacid that hybridizes to the Ysh1 nucleic acid (SEQ ID NO:76) or itscomplement under low stringency conditions, (xxxix) Yth1 (SEQ ID NO:77),or a mammalian homolog thereof, or a variant of Yth1 encoded by anucleic acid that hybridizes to the Yth1 nucleic acid (SEQ ID NO:78) orits complement under low stringency conditions, (xl) Tcl046w (SEQ IDNO:79), or a mammalian homolog thereof, or a variant of Ycl046w encodedby a nucleic acid that hybridizes to the Tcl046w nucleic acid (SEQ IDNO:80) or its complement under low stringency conditions, (xli) Ygr156w(SEQ ID NO:81), or a mammalian homolog thereof, or a variant of Ygr156wencoded by a nucleic acid that hybridizes to the Ygr156w nucleic acid(SEQ ID NO:82) or its complement under low stringency conditions, (xlii)Yhl035c (SEQ ID NO:83), or a mammalian homolog thereof, or a variant ofYhl035c encoded by a nucleic acid that hybridizes to the Yhl035c nucleicacid (SEQ ID NO:84) or its complement under low stringency conditions,(xliii) Ykl018w (SEQ ID NO:85), or a mammalian homolog thereof, or avariant of Ykl018w encoded by a nucleic acid that hybridizes to theYkl018w nucleic acid (SEQ ID NO:86) or its complement under lowstringency conditions, (xliv) Ylr221c (SEQ ID NO:87), or a mammalianhomolog thereof, or a variant of Ylr221c encoded by a nucleic acid thathybridizes to the Ylr221c nucleic acid (SEQ ID NO:88) or its complementunder low stringency conditions, (xlv) Ykl059c (SEQ ID NO:89), or amammalian homolog thereof, or a variant of Ykl059c encoded by a nucleicacid that hybridizes to the Ykl059c nucleic acid (SEQ ID NO:90) or itscomplement under low stringency conditions, (xlvi) Yml030w (SEQ IDNO:91), or a mammalian homolog thereof, or a variant of Yml030w encodedby a nucleic acid that hybridizes to the Yml030w nucleic acid (SEQ IDNO:92) or its complement under low stringency conditions, and (xlvii)Yor179c (SEQ ID NO:93), or a mammalian homolog thereof, or a variant ofYor179c encoded by a nucleic acid that hybridizes to the Yor179c nucleicacid (SEQ ID NO:94) or its complement under low stringency conditions,wherein said proteins are members of a native cellularPolyadenylation-complex, and wherein said low stringency conditionscomprise hybridization in a buffer comprising 35% formamide, 5×SSC, 50mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 55° C., andwashing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS for 1.5 hours at 60° C.
 4. The complex according toclaim 2, which comprises all but 1 of the 47 proteins.
 5. The complex ofclaim 1, 2, 3 or 4 comprising a functionally active derivative of saidfirst protein and/or a functionally active derivative of said secondprotein, wherein the functionally active derivative is a fusion proteincomprising said first protein or said second protein fused to an aminoacid sequence different from the first protein or second protein,respectively.
 6. The complex of claim 1, 2, 3 or 4 comprising a fragmentof said first protein and/or a fragment of said second protein, whichfragment binds to another protein component of said complex.
 7. Thecomplex of claim 1, 2, 3, 4, 5 or 6 that is involved in the 3′ endprocessing activity for mRNA.
 8. The complex of claim 5 wherein thefunctionally active derivative is a fusion protein comprising said firstprotein or said second protein fused to an affinity tag or label.
 9. Anantibody or a fragment of said antibody containing the binding domainthereof, which binds the complex of claim 1, 2, 3, 4, 5, 6 or 7 andwhich does not bind the first protein when uncomplexed or the secondprotein when uncomplexed.
 10. A process for processing RNA comprisingthe step of bringning into contact a product to any of claims 1-8 withRNA, such that the RNA is processed.
 11. A pharmaceutical compositioncomprising the protein complex of claim 1, 2, 3, 4, 5, 6, 7 or 8; and apharmaceutically acceptable carrier.
 12. A method for screening for amolecule that modulates directly or indirectly the function, activity,composition or formation of the complex of any one of claims 1-8comprising the steps of: (a) exposing said complex, or a cell ororganism containing said Polyadenylation-complex to one or morecandidate molecules; and (b) determining the amount of 3′ end processingactivity for mRNA of, or protein components of, said complex, wherein achange in said amount, activity, or protein components relative to saidamount, activity or protein components in the absence of said candidatemolecules indicates that the molecules modulate function, activity orcomposition of said complex.
 13. The method of claim 12, wherein theamount of said complex is determined.
 14. The method of claim 12,wherein the activity of said complex is determined.
 15. The method ofclaim 14, wherein said determining step comprises isolating from thecell or organism said Polyadenylation-complex to produce said isolatedcomplex and contacting said isolated complex with a RNA molecule suchthat the complex binds to the RNA.
 16. The method of claim 12, whereinthe protein components of said complex are determined.
 17. The method ofclaim 16, wherein said determining step comprises determining whether(i) Act1 (SEQ ID NO:1), or a mammalian homolog thereof, or a variant ofAct1 encoded by a nucleic acid that hybridizes to the Act1 nucleic acid(SEQ ID NO:2) or its complement under low stringency conditions, (ii)Cka1 (SEQ ID NO:7), or a mammalian homolog thereof, or a variant of Cka1encoded by a nucleic acid that hybridizes to the Cka1 nucleic acid (SEQID NO:8) or its complement under low stringency conditions, (iii) Eft2(SEQ ID NO:11), or a mammalian homolog thereof, or a variant of Eft2encoded by a nucleic acid that hybridizes to the Eft2 nucleic acid (SEQID NO:12) or its complement under low stringency conditions, (iv) Eno2(SEQ ID NO:13), or a mammalian homolog thereof, or a variant of Eno2encoded by a nucleic acid that hybridizes to the Eno2 nucleic acid (SEQID NO:14) or its complement under low stringency conditions, (v) Glc7(SEQ ID NO:15), or a mammalian homolog thereof, or a variant of Glc7encoded by a nucleic acid that hybridizes to the Glc7 nucleic acid (SEQID NO:16) or its complement under low stringency conditions, (vi) Gpm1(SEQ ID NO:17), or a mammalian homolog thereof, or a variant of Gpm1encoded by a nucleic acid that hybridizes to the Gpm1 nucleic acid (SEQID NO:18) or its complement under low stringency conditions, (vii) Hhf2(SEQ ID NO:21), or a mammalian homolog thereof, or a variant of Hhf2encoded by a nucleic acid that hybridizes to the Hhf2 nucleic acid (SEQID NO:22) or its complement under low stringency conditions, (viii) Hta1(SEQ ID NO:23), or a mammalian homolog thereof, or a variant of Hta1encoded by a nucleic acid that hybridizes to the Hta1 nucleic acid (SEQID NO:24) or its complement under low stringency conditions, (ix) Hsc82(SEQ ID NO:25), or a mammalian homolog thereof, or a variant of Hsc82encoded by a nucleic acid that hybridizes to the Hsc82 nucleic acid (SEQID NO:26) or its complement under low stringency conditions, (x) Imd2(SEQ ID NO:27), or a mammalian homolog thereof, or a variant of Imd2encoded by a nucleic acid that hybridizes to the Imd2 nucleic acid (SEQID NO:28) or its complement under low stringency conditions, (xi) Imd4(SEQ ID NO:29), or a mammalian homolog thereof, or a variant of Imd4encoded by a nucleic acid that hybridizes to the Imd4 nucleic acid (SEQID NO:30) or its complement under low stringency conditions, (xii) Met6(SEQ ID NO:31), or a mammalian homolog thereof, or a variant of Met6encoded by a nucleic acid that hybridizes to the Met6 nucleic acid (SEQID NO:32) or its complement under low stringency conditions, (xiii) Pdc1(SEQ ID NO:39), or a mammalian homolog thereof, or a variant of Pdc1encoded by a nucleic acid that hybridizes to the Pdc1 nucleic acid (SEQID NO:40) or its complement under low stringency conditions, (xiv) Pfk1(SEQ ID NO:41), or a mammalian homolog thereof, or a variant of Pfk1encoded by a nucleic acid that hybridizes to the Pfk1 nucleic acid (SEQID NO:42) or its complement under low stringency conditions, (xv) Ref2(SEQ ID NO:47), or a mammalian homolog thereof, or a variant of Ref2encoded by a nucleic acid that hybridizes to the Ref2 nucleic acid (SEQID NO:48) or its complement under low stringency conditions, (xvi) Sec13(SEQ ID NO:53), or a mammalian homolog thereof, or a variant of Sec13encoded by a nucleic acid that hybridizes to the Sec13 nucleic acid (SEQID NO:54) or its complement under low stringency conditions, (xvii)Sec31 (SEQ ID NO:55), or a mammalian homolog thereof, or a variant ofSec31 encoded by a nucleic acid that hybridizes to the Sec31 nucleicacid (SEQ ID NO:56) or its complement under low stringency conditions,(xviii) Ssa3 (SEQ ID NO:57), or a mammalian homolog thereof, or avariant of Ssa3 encoded by a nucleic acid that hybridizes to the Ssa3nucleic acid (SEQ ID NO:58) or its complement under low stringencyconditions, (xix) Ssu72 (SEQ ID NO:59), or a mammalian homolog thereof,or a variant of Ssu72 encoded by a nucleic acid that hybridizes to theSsu72 nucleic acid (SEQ ID NO:60) or its complement under low stringencyconditions, (xx) Taf60 (SEQ ID NO:61), or a mammalian homolog thereof,or a variant of Taf60 encoded by a nucleic acid that hybridizes to theTaf60 nucleic acid (SEQ ID NO:62) or its complement under low stringencyconditions, (xxi) Tkl1 (SEQ ID NO:65), or a mammalian homolog thereof,or a variant of Tkl1 encoded by a nucleic acid that hybridizes to theTkl1 nucleic acid (SEQ ID NO:66) or its complement under low stringencyconditions, (xxii) Tsa1 (SEQ ID NO:67), or a mammalian homolog thereof,or a variant of Tsa1 encoded by a nucleic acid that hybridizes to theTsa1 nucleic acid (SEQ ID NO:68) or its complement under low stringencyconditions, (xxiii) Tye7 (SEQ ID NO:69), or a mammalian homolog thereof,or a variant of Tye7 encoded by a nucleic acid that hybridizes to theTye7 nucleic acid (SEQ ID NO:70) or its complement under low stringencyconditions, (xxiv) Vid24 (SEQ ID NO:71), or a mammalian homolog thereof,or a variant of Vid24 encoded by a nucleic acid that hybridizes to theVid24 nucleic acid (SEQ ID NO:72) or its complement under low stringencyconditions, (xxv) Vps53 (SEQ ID NO:73), or a mammalian homolog thereof,or a variant of Vps53 encoded by a nucleic acid that hybridizes to theVps53 nucleic acid (SEQ ID NO:74) or its complement under low stringencyconditions, (xxvi) Ycl046w (SEQ ID NO:79), or a mammalian homologthereof, or a variant of Ycl046w encoded by a nucleic acid thathybridizes to the Ycl046w nucleic acid (SEQ ID NO:80) or its complementunder low stringency conditions, (xxvii) Ygr156w (SEQ ID NO:81), or amammalian homolog thereof, or a variant of Ygr156w encoded by a nucleicacid that hybridizes to the Ygr156w nucleic acid (SEQ ID NO:82) or itscomplement under low stringency conditions, (xxviii) Yhl035c (SEQ IDNO:83), or a mammalian homolog thereof, or a variant of Yhl035c encodedby a nucleic acid that hybridizes to the Yhl035c nucleic acid (SEQ IDNO:84) or its complement under low stringency conditions, (xxix) Ykl018w(SEQ ID NO:85), or a mammalian homolog thereof, or a variant of Ykl018wencoded by a nucleic acid that hybridizes to the Ykl018w nucleic acid(SEQ ID NO:86) or its complement under low stringency conditions, (xxx)Ylr221c (SEQ ID NO:87), or a mammalian homolog thereof, or a variant ofYlr221c encoded by a nucleic acid that hybridizes to the Ylr221c nucleicacid (SEQ ID NO:88) or its complement under low stringency conditions,(xxxi) Yml030w (SEQ ID NO:91), or a mammalian homolog thereof, or avariant of Yml030w encoded by a nucleic acid that hybridizes to theYml030w nucleic acid (SEQ ID NO:92) or its complement under lowstringency conditions, and (xxxii) Yor179c (SEQ ID NO:93), or amammalian homolog thereof, or a variant of Yor179c encoded by a nucleicacid that hybridizes to the Yor179c nucleic acid (SEQ ID NO:94) or itscomplement under low stringency conditions, is present in the complex,wherein said low stringency conditions comprise hybridization in abuffer comprising 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ug/ml denatured salmonsperm DNA, and 10% (wt/vol) dextran sulfate for 18-20 hours at 40° C.,washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS for 1.5 hours at 55° C., and washing in a bufferconsisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDSfor 1.5 hours at 60° C.
 18. The method of any of claim 12 to 17, whereinsaid method is a method of screening for a drug for treatment orprevention of a disease or disorder such as infectious diseases; viralinfections such as herpes simplex infections, Epstein-Barr-infections,influenza; metabolic disease such as metachromatic leukodystrophy;neurodegenerative disorders such as amyotrophic lateral sclerosis;cancer.
 19. A method for screening for a molecule that binds the complexof anyone of claim 1-8 comprising the following steps: (a) exposing saidcomplex, or a cell or organism containing said Polyadenylation-complex,to one or more candidate molecules; and (b) determining whether saidcomplex is bound by any of said candidate molecules.
 20. A method fordiagnosing or screening for the presence of a disease or disorder or apredisposition for developing a disease or disorder in a subject, whichdisease or disorder is characterized by an aberrant amount of 3′ endprocessing activity for mRNA of, or component composition of, thecomplex of any one of the claim 1-8, comprising determining the amountof, 3′ end processing activity for mRNA of, or protein components of,said complex in a sample derived from a subject, wherein a difference insaid amount, activity, or protein components of, said complex in ananalogous sample from a subject not having the disease or disorder orpredisposition indicates the presence in the subject of the disease ordisorder or predisposition.
 21. The method of claim 20, wherein theamount of said complex is determined.
 22. The method of claim 20,wherein the activity of said complex is determined.
 23. The method ofclaim 22, wherein said determining step comprises isolating from thesubject said Polyadenylation-complex to produce said isolated complexand contacting said isolated complex with a RNA molecule such that thecomplex binds to the RNA.
 24. The method of claim 20, wherein theprotein components of said complex are determined.
 25. The method ofclaim 24, wherein said determining step comprises determining whether(i) Act1 (SEQ ID NO:1), or a mammalian homolog thereof, or a variant ofAct1 encoded by a nucleic acid that hybridizes to the Act1 nucleic acid(SEQ ID NO:2) or its complement under low stringency conditions, (ii)Cka1 (SEQ ID NO:7), or a mammalian homolog thereof, or a variant of Cka1encoded by a nucleic acid that hybridizes to the Cka1 nucleic acid (SEQID NO:8) or its complement under low stringency conditions, (iii) Eft2(SEQ ID NO:11), or a mammalian homolog thereof, or a variant of Eft2encoded by a nucleic acid that hybridizes to the Eft2 nucleic acid (SEQID NO:12) or its complement under low stringency conditions, (iv) Eno2(SEQ ID NO:13), or a mammalian homolog thereof, or a variant of Eno2encoded by a nucleic acid that hybridizes to the Eno2 nucleic acid (SEQID NO:14) or its complement under low stringency conditions, (v) Glc7(SEQ ID NO:15), or a mammalian homolog thereof, or a variant of Glc7encoded by a nucleic acid that hybridizes to the Glc7 nucleic acid (SEQID NO:16) or its complement under low stringency conditions, (vi) Gpm1(SEQ ID NO:17), or a mammalian homolog thereof, or a variant of Gpm1encoded by a nucleic acid that hybridizes to the Gpm1 nucleic acid (SEQID NO:18) or its complement under low stringency conditions, (vii) Hhf2(SEQ ID NO:21), or a mammalian homolog thereof, or a variant of Hhf2encoded by a nucleic acid that hybridizes to the Hhf2 nucleic acid (SEQID NO:22) or its complement under low stringency conditions, (viii) Hta1(SEQ ID NO:23), or a mammalian homolog thereof, or a variant of Hta1encoded by a nucleic acid that hybridizes to the Hta1 nucleic acid (SEQID NO:24) or its complement under low stringency conditions, (ix) Hsc82(SEQ ID NO:25), or a mammalian homolog thereof, or a variant of Hsc82encoded by a nucleic acid that hybridizes to the Hsc82 nucleic acid (SEQID NO:26) or its complement under low stringency conditions, (x) Imd2(SEQ ID NO:27), or a mammalian homolog thereof, or a variant of Imd2encoded by a nucleic acid that hybridizes to the Imd2 nucleic acid (SEQID NO:28) or its complement under low stringency conditions, (xi) Imd4(SEQ ID NO:29), or a mammalian homolog thereof, or a variant of Imd4encoded by a nucleic acid that hybridizes to the Imd4 nucleic acid (SEQID NO:30) or its complement under low stringency conditions, (xii) Met6(SEQ ID NO:31), or a mammalian homolog thereof, or a variant of Met6encoded by a nucleic acid that hybridizes to the Met6 nucleic acid (SEQID NO:32) or its complement under low stringency conditions, (xiii) Pdc1(SEQ ID NO:39), or a mammalian homolog thereof, or a variant of Pdc1encoded by a nucleic acid that hybridizes to the Pdc1 nucleic acid (SEQID NO:40) or its complement under low stringency conditions, (xiv) Pfk1(SEQ ID NO:41), or a mammalian homolog thereof, or a variant of Pfk1encoded by a nucleic acid that hybridizes to the Pfk1 nucleic acid (SEQID NO:42) or its complement under low stringency conditions, (xv) Ref2(SEQ ID NO:47), or a mammalian homolog thereof, or a variant of Ref2encoded by a nucleic acid that hybridizes to the Ref2 nucleic acid (SEQID NO:48) or its complement under low stringency conditions, (xvi) Sec13(SEQ ID NO:53), or a mammalian homolog thereof, or a variant of Sec13encoded by a nucleic acid that hybridizes to the Sec13 nucleic acid (SEQID NO:54) or its complement under low stringency conditions, (xvii)Sec31 (SEQ ID NO:55), or a mammalian homolog thereof, or a variant ofSec31 encoded by a nucleic acid that hybridizes to the Sec31 nucleicacid (SEQ ID NO:56) or its complement under low stringency conditions,(xviii) Ssa3 (SEQ ID NO:57), or a mammalian homolog thereof, or avariant of Ssa3 encoded by a nucleic acid that hybridizes to the Ssa3nucleic acid (SEQ ID NO:58) or its complement under low stringencyconditions, (xix) Ssu72 (SEQ ID NO:59), or a mammalian homolog thereof,or a variant of Ssu72 encoded by a nucleic acid that hybridizes to theSsu72 nucleic acid (SEQ ID NO:60) or its complement under low stringencyconditions, (xx) Taf60 (SEQ ID NO:61), or a mammalian homolog thereof,or a variant of Taf60 encoded by a nucleic acid that hybridizes to theTaf60 nucleic acid (SEQ ID NO:62) or its complement under low stringencyconditions, (xxi) Tkl1 (SEQ ID NO:65), or a mammalian homolog thereof,or a variant of Tkl1 encoded by a nucleic acid that hybridizes to theTkl1 nucleic acid (SEQ ID NO:66) or its complement under low stringencyconditions, (xxii) Tsa1 (SEQ ID NO:67), or a mammalian homolog thereof,or a variant of Tsa1 encoded by a nucleic acid that hybridizes to theTsa1 nucleic acid (SEQ ID NO:68) or its complement under low stringencyconditions, (xxiii) Tye7 (SEQ ID NO:69), or a mammalian homolog thereof,or a variant of Tye7 encoded by a nucleic acid that hybridizes to theTye7 nucleic acid (SEQ ID NO:70) or its complement under low stringencyconditions, (xxiv) Vid24 (SEQ ID NO:71), or a mammalian homolog thereof,or a variant of Vid24 encoded by a nucleic acid that hybridizes to theVid24 nucleic acid (SEQ ID NO:72) or its complement under low stringencyconditions, (xxv) Vps53 (SEQ ID NO:73), or a mammalian homolog thereof,or a variant of Vps53 encoded by a nucleic acid that hybridizes to theVps53 nucleic acid (SEQ ID NO:74) or its complement under low stringencyconditions, (xxvi) Tcl046w (SEQ ID NO:79), or a mammalian homologthereof, or a variant of Tcl046w encoded by a nucleic acid thathybridizes to the Tcl046w nucleic acid (SEQ ID NO:80) or its complementunder low stringency conditions, (xxvii) Ygr156w (SEQ ID NO:81), or amammalian homolog thereof, or a variant of Ygr156w encoded by a nucleicacid that hybridizes to the Ygr156w nucleic acid (SEQ ID NO:82) or itscomplement under low stringency conditions, (xxviii) Yhl035c (SEQ IDNO:83), or a mammalian homolog thereof, or a variant of Yhl035c encodedby a nucleic acid that hybridizes to the Yhl035c nucleic acid (SEQ IDNO:84) or its complement under low stringency conditions, (xxix) Ykl018w(SEQ ID NO:85), or a mammalian homolog thereof, or a variant of Ykl018wencoded by a nucleic acid that hybridizes to the Ykl018w nucleic acid(SEQ ID NO:86) or its complement under low stringency conditions, (xxx)Ylr221c (SEQ ID NO:87), or a mammalian homolog thereof, or a variant ofYlr221c encoded by a nucleic acid that hybridizes to the Ylr221c nucleicacid (SEQ ID NO:88) or its complement under low stringency conditions,(xxxi) Yml030w (SEQ ID NO:91), or a mammalian homolog thereof, or avariant of Yml030w encoded by a nucleic acid that hybridizes to theYml030w nucleic acid (SEQ ID NO:92) or its complement under lowstringency conditions, and (xxxii) Yor179c (SEQ ID NO:93), or amammalian homolog thereof, or a variant of Yor179c encoded by a nucleicacid that hybridizes to the Yor179c nucleic acid (SEQ ID NO:94) or itscomplement under low stringency conditions, is present in the complex,wherein said low stringency conditions comprise hybridization in abuffer comprising 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ug/ml denatured salmonsperm DNA, and 10% (wt/vol) dextran sulfate for 18-20 hours at 40° C.,washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS for 1.5 hours at 55° C., and washing in a bufferconsisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDSfor 1.5 hours at 60° C.
 26. A method for treating or preventing adisease or disorder characterized by an aberrant amount of, 3′ endprocessing activity for mRNA of, or component composition of, thecomplex of anyone of claim 1-8, comprising administering to a subject inneed of such treatment or prevention a therapeutically effective amountof one or more molecules that modulate the amount of, 3′ end processingactivity for mRNA of, or protein components of, said complex.
 27. Themethod according to claim 26, wherein said disease or disorder involvesdecreased levels of the amount or activity of said complex.
 28. Themethod according to claim 26, wherein said disease or disorder involvesincreased levels of the amount or activity of said complex.
 29. Use of amolecule that modulates the amount of, 3′ end processing activity formRNA of, or the protein components of the complex of any one of claim1-8 for the manufacture of a medicament for the treatment or preventionof a disease or disorder such as infectious diseases; viral infectionssuch as herpes simplex infections, Epstein-Barr-infections, influenza;metabolic disease such as metachromatic leukodystrophy;neurodegenerative disorders such as amyotrophic lateral sclerosis;cancer.
 30. A kit comprising in one or more containers (a) an isolatedfirst protein, or a functionally active fragment or functionally activederivative thereof, which first protein is selected from the groupconsisting of: (i) Cft1 (SEQ ID NO:3), or a mammalian homolog thereof,or a variant of Cft1 encoded by a nucleic acid that hybridizes to theCft1 nucleic acid (SEQ ID NO:4) or its complement under low stringencyconditions, (ii) Cft2 (SEQ ID NO:5), or a mammalian homolog thereof, ora variant of Cft2 encoded by a nucleic acid that hybridizes to the Cft2nucleic acid (SEQ ID NO:6) or its complement under low stringencyconditions, (iii) Clp1 (SEQ ID NO:9), or a mammalian homolog thereof, ora variant of Clp1 encoded by a nucleic acid that hybridizes to the Clp1nucleic acid (SEQ ID NO:10) or its complement under low stringencyconditions, (iv) Fip1 (SEQ ID NO:19), or a mammalian homolog thereof, ora variant of Fip1 encoded by a nucleic acid that hybridizes to the Fip1nucleic acid (SEQ ID NO:20) or its complement under low stringencyconditions, (v) Pab1 (SEQ ID NO:33), or a mammalian homolog thereof, ora variant of Pab1 encoded by a nucleic acid that hybridizes to the Pab1nucleic acid (SEQ ID NO:34) or its complement under low stringencyconditions, (vi) Pap1 (SEQ ID NO:35), or a mammalian homolog thereof, ora variant of Pap1 encoded by a nucleic acid that hybridizes to the Pap1nucleic acid (SEQ ID NO:36) or its complement under low stringencyconditions, (vii) Pcf11 (SEQ ID NO:37), or a mammalian homolog thereof,or a variant of Pcf11 encoded by a nucleic acid that hybridizes to thePcf11 nucleic acid (SEQ ID NO:38) or its complement under low stringencyconditions, (viii) Pfs2 (SEQ ID NO:43), or a mammalian homolog thereof,or a variant of Pfs2 encoded by a nucleic acid that hybridizes to thePfs2 nucleic acid (SEQ ID NO:44) or its complement under low stringencyconditions, (ix) Pta1 (SEQ ID NO:45), or a mammalian homolog thereof, ora variant of Pta1 encoded by a nucleic acid that hybridizes to the Pta1nucleic acid (SEQ ID NO:46) or its complement under low stringencyconditions, (x) Rna14 (SEQ ID NO:49), or a mammalian homolog thereof, ora variant of Rna14 encoded by a nucleic acid that hybridizes to theRna14 nucleic acid (SEQ ID NO:50) or its complement under low stringencyconditions, (xi) Rna15 (SEQ ID NO:51), or a mammalian homolog thereof,or a variant of Rna15 encoded by a nucleic acid that hybridizes to theRna15 nucleic acid (SEQ ID NO:52) or its complement under low stringencyconditions, (xii) Tif4632 (SEQ ID NO:63), or a mammalian homologthereof, or a variant of Tif4632 encoded by a nucleic acid thathybridizes to the Tif4632 nucleic acid (SEQ ID NO:64) or its complementunder low stringency conditions, (xiii) Ykl059c (SEQ ID NO:89), or amammalian homolog thereof, or a variant of Ykl059c encoded by a nucleicacid that hybridizes to the Ykl059c nucleic acid (SEQ ID NO:90) or itscomplement under low stringency conditions, (xiv) Ysh1 (SEQ ID NO:75),or a mammalian homolog thereof, or a variant of Ysh1 encoded by anucleic acid that hybridizes to the Ysh1 nucleic acid (SEQ ID NO:76) orits complement under low stringency conditions, and (xv) Yth1 (SEQ IDNO:77), or a mammalian homolog thereof, or a variant of Yth1 encoded bya nucleic acid that hybridizes to the Yth1 nucleic acid (SEQ ID NO:78)or its complement under low stringency conditions; and (b) a secondprotein, or a functionally active fragment or functionally activederivative thereof, which second protein is selected from the groupconsisting of: (i) Act1 (SEQ ID NO:1), or a mammalian homolog thereof,or a variant of Act1 encoded by a nucleic acid that hybridizes to theAct1 nucleic acid (SEQ ID NO:2) or its complement under low stringencyconditions, (ii) Cka1 (SEQ ID NO:7), or a mammalian homolog thereof, ora variant of Cka1 encoded by a nucleic acid that hybridizes to the Cka1nucleic acid (SEQ ID NO:8) or its complement under low stringencyconditions, (iii) Eft2 (SEQ ID NO:11), or a mammalian homolog thereof,or a variant of Eft2 encoded by a nucleic acid that hybridizes to theEft2 nucleic acid (SEQ ID NO:12) or its complement under low stringencyconditions, (iv) Eno2 (SEQ ID NO:13), or a mammalian homolog thereof, ora variant of Eno2 encoded by a nucleic acid that hybridizes to the Eno2nucleic acid (SEQ ID NO:14) or its complement under low stringencyconditions, (v) Glc7 (SEQ ID NO:15), or a mammalian homolog thereof, ora variant of Glc7 encoded by a nucleic acid that hybridizes to the Glc7nucleic acid (SEQ ID NO:16) or its complement under low stringencyconditions, (vi) Gpm1 (SEQ ID NO:17), or a mammalian homolog thereof, ora variant of Gpm1 encoded by a nucleic acid that hybridizes to the Gpm1nucleic acid (SEQ ID NO:18) or its complement under low stringencyconditions, (vii) Hhf2 (SEQ ID NO:21), or a mammalian homolog thereof,or a variant of Hhf2 encoded by a nucleic acid that hybridizes to theHhf2 nucleic acid (SEQ ID NO:22) or its complement under low stringencyconditions, (viii) Hta1 (SEQ ID NO:23), or a mammalian homolog thereof,or a variant of Hta1 encoded by a nucleic acid that hybridizes to theHta1 nucleic acid (SEQ ID NO:24) or its complement under low stringencyconditions, (ix) Hsc82 (SEQ ID NO:25), or a mammalian homolog thereof,or a variant of Hsc82 encoded by a nucleic acid that hybridizes to theHsc82 nucleic acid (SEQ ID NO:26) or its complement under low stringencyconditions, (x) Imd2 (SEQ ID NO:27), or a mammalian homolog thereof, ora variant of Imd2 encoded by a nucleic acid that hybridizes to the Imd2nucleic acid (SEQ ID NO:28) or its complement under low stringencyconditions, (xi) Imd4 (SEQ ID NO:29), or a mammalian homolog thereof, ora variant of Imd4 encoded by a nucleic acid that hybridizes to the Imd4nucleic acid (SEQ ID NO:30) or its complement under low stringencyconditions, (xii) Met6 (SEQ ID NO:31), or a mammalian homolog thereof,or a variant of Met6 encoded by a nucleic acid that hybridizes to theMet6 nucleic acid (SEQ ID NO:32) or its complement under low stringencyconditions, (xiii) Pdc1 (SEQ ID NO:39), or a mammalian homolog thereof,or a variant of Pdc1 encoded by a nucleic acid that hybridizes to thePdc1 nucleic acid (SEQ ID NO:40) or its complement under low stringencyconditions, (xiv) Pfk1 (SEQ ID NO:41), or a mammalian homolog thereof,or a variant of Pfk1 encoded by a nucleic acid that hybridizes to thePfk1 nucleic acid (SEQ ID NO:42) or its complement under low stringencyconditions, (xv) Ref2 (SEQ ID NO:47), or a mammalian homolog thereof, ora variant of Ref2 encoded by a nucleic acid that hybridizes to the Ref2nucleic acid (SEQ ID NO:48) or its complement under low stringencyconditions, (xvi) Sec13 (SEQ ID NO:53), or a mammalian homolog thereof,or a variant of Sec13 encoded by a nucleic acid that hybridizes to theSec13 nucleic acid (SEQ ID NO:54) or its complement under low stringencyconditions, (xvii) Sec31 (SEQ ID NO:55), or a mammalian homolog thereof,or a variant of Sec31 encoded by a nucleic acid that hybridizes to theSec31 nucleic acid (SEQ ID NO:56) or its complement under low stringencyconditions, (xviii) Ssa3 (SEQ ID NO:57), or a mammalian homolog thereof,or a variant of Ssa3 encoded by a nucleic acid that hybridizes to theSsa3 nucleic acid (SEQ ID NO:58) or its complement under low stringencyconditions, (xix) Ssu72 (SEQ ID NO:59), or a mammalian homolog thereof,or a variant of Ssu72 encoded by a nucleic acid that hybridizes to theSsu72 nucleic acid (SEQ ID NO:60) or its complement under low stringencyconditions, (xx) Taf60 (SEQ ID NO:61), or a mammalian homolog thereof,or a variant of Taf60 encoded by a nucleic acid that hybridizes to theTaf60 nucleic acid (SEQ ID NO:62) or its complement under low stringencyconditions, (xxi) Tkl1 (SEQ ID NO:65), or a mammalian homolog thereof,or a variant of Tkl1 encoded by a nucleic acid that hybridizes to theTkl1 nucleic acid (SEQ ID NO:66) or its complement under low stringencyconditions, (xxii) Tsa1 (SEQ ID NO:67), or a mammalian homolog thereof,or a variant of Tsa1 encoded by a nucleic acid that hybridizes to theTsa1 nucleic acid (SEQ ID NO:68) or its complement under low stringencyconditions, (xxiii) Tye7 (SEQ ID NO:69), or a mammalian homolog thereof,or a variant of Tye7 encoded by a nucleic acid that hybridizes to theTye7 nucleic acid (SEQ ID NO:70) or its complement under low stringencyconditions, (xxiv) Vid24 (SEQ ID NO:71), or a mammalian homolog thereof,or a variant of Vid24 encoded by a nucleic acid that hybridizes to theVid24 nucleic acid (SEQ ID NO:72) or its complement under low stringencyconditions, (xxv) Vps53 (SEQ ID NO:73), or a mammalian homolog thereof,or a variant of Vps53 encoded by a nucleic acid that hybridizes to theVps53 nucleic acid (SEQ ID NO:74) or its complement under low stringencyconditions, (xxvi) Ycl046w (SEQ ID NO:79), or a mammalian homologthereof, or a variant of Ycl046w encoded by a nucleic acid thathybridizes to the Ycl046w nucleic acid (SEQ ID NO:80) or its complementunder low stringency conditions, (xxvii) Ygr156w (SEQ ID NO:81), or amammalian homolog thereof, or a variant of Ygr156w encoded by a nucleicacid that hybridizes to the Ygr156w nucleic acid (SEQ ID NO:82) or itscomplement under low stringency conditions, (xxviii) Yhl035c (SEQ IDNO:83), or a mammalian homolog thereof, or a variant of Yhl035c encodedby a nucleic acid that hybridizes to the Yhl035c nucleic acid (SEQ IDNO:84) or its complement under low stringency conditions, (xxix) Ykl018w(SEQ ID NO:85), or a mammalian homolog thereof, or a variant of Ykl018wencoded by a nucleic acid that hybridizes to the Ykl018w nucleic acid(SEQ ID NO:86) or its complement under low stringency conditions, (xxx)Ylr221c (SEQ ID NO:87), or a mammalian homolog thereof, or a variant ofYlr221c encoded by a nucleic acid that hybridizes to the Ylr221c nucleicacid (SEQ ID NO:88) or its complement under low stringency conditions,(xxxi) Yml030w (SEQ ID NO:91), or a mammalian homolog thereof, or avariant of Yml030w encoded by a nucleic acid that hybridizes to theYml030w nucleic acid (SEQ ID NO:92) or its complement under lowstringency conditions, and (xxxii) Yor179c (SEQ ID NO:93), or amammalian homolog thereof, or a variant of Yor179c encoded by a nucleicacid that hybridizes to the Yor179c nucleic acid (SEQ ID NO:94) or itscomplement under low stringency conditions, wherein said first proteinand said second protein are members of a native cellularPolyadenylation-complex, and wherein said low stringency conditionscomprise hybridization in a buffer comprising 35% formamide, 5×SSC, 50mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 55° C., andwashing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS for 1.5 hours at 60° C.
 31. A kit comprising in acontainer the isolated complex of any one of claim 1-8 or the antibodyof claim
 9. 32. A kit for processing RNA comprising in a container theisolated complex of any of claims 1-8 optionally together with furthercomponents such as reagents and working instructions.
 33. A kit for thediagnosis of a disease of mammals, preferentially for a disease ordisorder such as infectious diseases; viral infections such as herpessimplex infections, Epstein-Barr-infections, influenza; metabolicdisease such as metachromatic leukodystrophy; neurodegenerativedisorders such as amyotrophic lateral sclerosis or cancer, comprising aproduct according to any of the claims 1-8 optionally together withfurther components such as reagents and working instructions.
 34. Thecomplex of any one of claim 1-8, or the antibody or fragment of claim 9,for use in a method of diagnosing a disease or disorder such asinfectious diseases; viral infections such as herpes simplex infections,Epstein-Barr-infections, influenza; metabolic disease such asmetachromatic leukodystrophy; neurodegenerative disorders such asamyotrophic lateral sclerosis; cancer.
 35. A method for the productionof a pharmaceutical composition comprising carrying out the method ofclaim 12 or 19 to identify a molecule that modulates the function,activity, composition or formation of said complex, and furthercomprising mixing the identified molecule with a pharmaceuticallyacceptable carrier.
 36. A process for preparing complex of claim 1-8 andoptionally the components thereof comprising the following steps:expressing such a protein in a target cell, isolating the proteincomplex which is attached to the tagged protein, and optionallydisassociating the protein complex and isolating the individual complexmembers.
 37. The process according to claim 36 characterized in that thetagged protein comprises two different tags which allow two separateaffinity purification steps.
 38. The process according to any of claim36-37 characterized in that two tags are separated by a cleavage sitefor a protease.
 39. Component of the Polyadenylation-complex obtainableby a process according to any of claim 36-38.
 40. Complex of claim 1-8and/or protein thereof as a target for an active agent of apharmaceutical, preferably a drug target in the treatment or preventionof a disease or disorder such as infectious diseases; viral infectionssuch as herpes simplex infections, Epstein-Barr-infections, influenza;metabolic disease such as metachromatic leukodystrophy;neurodegenerative disorders such as amyotrophic lateral sclerosis;cancer.
 41. Component of the Polyadenylation-complex selected from a)yeast proteins (i) Tcl046w (SEQ ID NO:59), (ii) Ygr156w (SEQ ID NO:61),(iii) Yhl035c (SEQ ID NO:63), (iv) Ykl018w (SEQ ID NO:179), (v) Ylr221c(SEQ ID NO:67), (vi) Yml030w (SEQ ID NO:69), and (vii) Yor179c (SEQ IDNO:71). b) the mammalian homologs/orthologs of the proteins of (a), andc) a functionally active fragment or functionally active derivate of theproteins according to (a) and (b) carrying one or more amino acidsubstitutions, deletions and/or additions.
 42. Component as described inclaim 41, characterized in that it is encoded by a nucleic acid sequencewhich hybridizes to a nucleic acid sequence encoding any of the yeastproteins listed in claim 41 under low stringency conditions, whereinsaid low stringency conditions comprise hybridization in a buffercomprising 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA,0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ug/ml denatured salmon sperm DNA,and 10% (wt/vol) dextran sulfate for 18-20 hours at 40° C., washing in abuffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1%SDS for 1.5 hours at 55° C., and washing in a buffer consisting of2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at60° C.
 43. Nucleic acid encoding a component to any of claims 41 and 42.44. Construct, preferably a vector construct, comprising (a) a nucleicacid according to claim 41 and at least one further nucleic acid whichis normally not associated with the nucleic acid according to claim 43,or (b) at least two separate nucleic acid sequences each encoding adifferent protein, or a functionally active fragment or a functionallyactive derivative thereof at least one of said proteins, or functionallyactive fragments or functionally active derivative thereof selected fromthe first group of proteins according to claim 1 (a) and at least one ofsaid proteins, or functionally active fragments or functionally activederivative thereof selected from the second group of proteins accordingto claim 1 (b).
 45. Host cell containing a nucleic acid of claim 43and/or a construct of claim 44 or containing several vectors comprisingon different vectors the nucleic acid sequence encoding at least one ofthe proteins, or functionally active fragments or functionally activederivatives thereof selected from the first group of proteins accordingto claim 1 (a) and at least one of the proteins, or functionally activefragments or functionally active derivatives thereof selected from thesecond group of proteins according to claim 1 (b).